Photosensitizing antibody-fluorophore conjugates

ABSTRACT

The present disclosure relates to compositions and methods of killing cells. In particular examples, the method includes contacting a cell having a cell surface protein with a therapeutically effective amount of an antibody-IR700 molecule, wherein the antibody specifically binds to the cell surface protein, such as a tumor-specific antigen on the surface of a tumor cell. The cell is subsequently irradiated, such as at a wavelength of 660 to 740 nm at a dose of at least 1 J cm−2. The cell is also contacted with one or more therapeutic agents (such as an anti-cancer agent), for example about 0 to 8 hours after irradiating the cell, thereby killing the cell. Also provided are methods of imaging cell killing in real time, using fluorescence lifetime imaging. Also provided are wearable devices that include an article of clothing, jewelry, or covering; and an NIR LED incorporated into the article, which can be used with the disclosed methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/868,040 filedSep. 28, 2015, which is a divisional of U.S. Ser. No. 14/126,060 filedDec. 13, 2013, now U.S. Pat. No. 9,358,306, which is the U.S. NationalStage of International Application No. PCT/US2012/044421, filed Jun. 27,2012, which was published in English under PCT Article 21(2), which is acontinuation-in-part of U.S. application Ser. No. 13/180,111 filed Jul.11, 2011, now U.S. Pat. No. 8,524,239, which claims priority to U.S.Provisional Application No. 61/363,079, filed Jul. 9, 2010, all hereinincorporated by reference.

FIELD OF THE DISCLOSURE

This application relates to antibody-IR700 conjugates, and methods oftheir use to kill cells that specifically bind to the antibody followingirradiation with infrared (NIR) light. Also provided are devices thatincorporate NIR light emitting diodes (LEDs) that can also be used withthe disclosed conjugates and methods.

BACKGROUND

Cancer was responsible for about 13% of all human deaths in 2007.Although there are several therapies for cancer, there remains a needfor therapies that effectively kill the tumor cells while not harmingnon-cancerous cells.

In order to minimize the side effects of conventional cancer therapies,including surgery, radiation and chemotherapy, molecular targeted cancertherapies have been developed. Among the existing targeted therapies,monoclonal antibodies (MAb) therapy have the longest history, and todate, over 25 therapeutic MAbs have been approved by the Food and DrugAdministration (FDA) (Waldmann, Nat Med 9:269-277, 2003); Reichert etal., Nat Biotechnol 23:1073-1078, 2005). Effective MAb therapytraditionally depends on three mechanisms: antibody-dependent cellularcytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), andreceptor blockade and requires multiple high doses of the MAb. MAbs havealso been used at lower doses as vectors to deliver therapies such asradionuclides (Goldenberg et al., J Clin Oncol 24, 823-834, 2006) orchemical or biological toxins (Pastan et al., Nat Rev Cancer 6:559-565,2006). Ultimately, however, dose limiting toxicity relates to thebiodistribution and catabolism of the antibody conjugates.

Conventional photodynamic therapy (PDT), which combines aphotosensitizing agent with the physical energy of non-ionizing light tokill cells, has been less commonly employed for cancer therapy becausethe current non-targeted photosensitizers are also taken up in normaltissues, thus, causing serious side effects, although the excitationlight itself is harmless in the near infrared (NIR) range (FIG. 9).

SUMMARY OF THE DISCLOSURE

Provided herein are antibody-IR700 molecules and methods of their usefor killing a target cell, such as a tumor cell. In particular examplesthe methods are specific in that non-target cells, such as normal cells,are not killed in significant numbers (such as less than 1% of normalcells are killed), but the target cells are. In particular examples themethod includes contacting a cell having a cell surface protein with atherapeutically effective amount of an antibody-IR700 molecule, whereinthe antibody (or other specific binding agent) specifically binds to thecell surface protein. Specific non-limiting examples of antibody-IR700molecules include Panitumumab-IR700, Trastuzumab-IR700, andHuJ591-IR700. The cell is irradiated at a wavelength of 660 to 740 nm,such as 660 to 710 nm (for example, 680 nm) at a dose of at least 1 Jcm⁻² (such as at least 50 J cm⁻²). The method also includes contactingthe cell with one or more therapeutic agents (such as an anti-canceragent), for example within about 8 hours after irradiating the cell,thereby killing the cell. Such methods can further include detecting thecell with fluorescence lifetime imaging (FLT), for example about 0 to 48hours after irradiating the cell, thereby permitting detection of cellkilling in real-time.

Also provided are methods of detecting cell killing in real-time. Suchmethods can include contacting a cell comprising a cell surface proteinwith a therapeutically effective amount of one or more antibody-IR700molecules as described above, irradiating the cell at a wavelength of660 to 740 nm and at a dose of at least 30 J cm⁻² (such as a dosesufficient to shorten IR700 FLT by at least 25% for example 30 to 50 Jcm⁻²), and detecting the cell with fluorescence lifetime imaging about 0to 48 hours (such as at least 6 hours) after irradiating the cell,thereby detecting the cell killing in real-time.

Any target cell can be killed (and in some examples detected inreal-time) with the disclosed antibody-IR700 molecules and methods, forexample by using one or more antibodies that binds to one or moreproteins on the target cell surface (such as a receptor), wherein theprotein(s) on the target cell surface is not significantly found onnon-target cells (such as normal healthy cells) and thus the antibodywill not significantly bind to the non-target cells. In one example thecell surface protein is a tumor-specific protein, such as HER1, HER2, orPSMA.

In some examples, the cell to be killed is present in a subject. In suchexamples, the method can include administering a therapeuticallyeffective amount of the antibody-IR700 molecule to the subject andirradiating the subject, for example irradiating a tumor in the subject.In some examples, the method can further include selecting a subjectwith a tumor that expresses a cell surface protein that can specificallybind to the antibody-IR700 molecule.

Also provided are devices, such as those that can be worn by a patient.Such devices can include an article of clothing, jewelry, or a covering,and a near infrared (NIR) light emitting diode (LED) that isincorporated into the article of clothing, jewelry, or covering. Suchdevices can further include power and/or cooling sources. This permitsthe patient to wear the device (or be covered by the device) forextended periods of time, thus permitting treatment of tumor cellspresent in the blood or circulatory system. Methods of using the deviceare also provided.

The foregoing and other features of the disclosure will become moreapparent from the following detailed description of a severalembodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A is a digital image showing the labeling of cells with aTrastuzumab-IR700 conjugate (Tra-IR700) at 4° C. for 1 hour or 37° C.for 6 hours. Light images also shown. Scale bar, 30 μm.

FIG. 1B is a digital image showing the lysosomal localization ofTra-1R700 6 h after incubation. Scale bar, 50 μm.

FIG. 1C is a digital image showing is a digital image showing before andafter incubation with Tra-IR700 at 37° C. for 6 hours followed byphotoimmunotherapy (PIT). Scale bar, 50 μm.

FIG. 1D is a bar graph showing the irradiation dose dependent and targetspecific cell death in response to Tra-1R700 mediated PIT. Data aremeans±s.e.m. (n=at least 4, *** P<0.001 vs. non treatment control,Student's t test).

FIG. 1E is a bar graph showing the long term growth inhibition inresponse to Tra-1R700 mediated PIT. Data are means±s.e.m. (n=3, **P<0.01 vs. non treatment control, Student's t test).

FIG. 1F is a digital image showing the microscopic observation of growthinhibition in response to TraIR700 mediated PIT. Scale bar, 100 μm

FIG. 1G is a bar graph showing that internalization of Tra-1R700 was notrequired for phototoxic cell death. Data are means±s.e.m. (n=3).

FIG. 1H is a bar graph showing that target specific membrane binding ofTra-1R700 only induced phototoxic cell death. Data are means±s.e.m.(n=3).

FIG. 1I is a graph showing that HER2 negatively expressing A431 cellsdid not show phototoxic effects with Tra-1R700 mediated PIT (n=3).

FIG. 1J is a bar graph showing sodium azide (NaN₃) concentrationdependent inhibition of phototoxic cell death induced by Tra-1R700mediated PIT. Data are means±s.e.m. (n=3, *** P<0.001, ** P<0.01 vs. 2.0J cm-2 PIT treatment without NaN3 control, Student's t test). DIC:differential interference contrast. PanIR: Pan-1R700.

FIG. 2A is a graph showing that long term growth inhibition was notobserved in Balb/3T3 (HER2 negative) cells treated with Tra-1R700(TraIR) and exposed to light. Data are means±s.e.m. (n=3).

FIG. 2B is a digital image showing that Free IR700 dye did notincorporate into 3T3/HER2 cells. Fluorescence image was taken withoutwashing the cells. Cells were darker than the media containing freeIR700 dye. Scale bar, 50 μm.

FIG. 2C is a graph showing that TraIR700 mediated phototoxicity wasdose-dependently blocked by the excess of unconjugated trastuzumab(Tra). Data are means±s.e.m. (n=3).

FIG. 2D is a graph showing that Tra-1R700 binding for 3T3/HER2 cells wasblocked by unconjugated trastuzumab dose-dependent manner (n=3). DIC:differential interference contrast FIG. 3A is a digital image showingthat induction of target specific photoimmunotherapy (PIT) lead to HER2expressing cell specific necrotic cell death. Scale bar, 50 μm.

FIG. 3B is a digital image showing that HER2 specific cell death wasconfirmed with fluorescence microscopy with LIVE/DEAD Green staining.Scale bar, 100 μm.

FIG. 3C is a plot showing flow cytometric analysis for detectingHER2-specific cell death induced by Tra-1R700 (TraIR) mediated PIT.Upper left quadrant: TraIR700 positive, live cells; upper rightquadrant; Tra-1R700 positive, dead cells; lower left quadrant: Tra-1R700negative, live cells; lower right quadrant: Tra-1R700 negative, deadcells (n=3). DIC: differential interference contrast.

FIG. 4A is a digital image showing the biodistribution of Tra-1R700.3T3/HER2 tumors (both sides of dorsum) were visualized with IR700fluorescence as early as 1 day after Tra-1R700 injection (300 μg). Rightside of the tumor was irradiated with near infrared (NIR) light on day1, while left side of the tumor was covered with black tape. Tumorshrinkage was confirmed on day 7. Dashed line: irradiated tumor, solidline: non-irradiated tumor. No other specific localization of IR700 wasfound except for the bladder accumulation on day 1 due to the excretionof unbound dye (n=5 mice).

FIG. 4B is a graph showing mean tumor volume following administration invivo of Tra-IR700 or carrier alone followed by PIT (50 J cm⁻²). Data aremeans±s.e.m. (at least n=12 mice in each group, *** P<0.001,” P<0.01 vs.non treatment control, Kruskal-Wallis test with post-test). Tra:trastuzumab.

FIG. 5A is a digital image showing a microscopic observation of beforeand after Pan-1R700 mediated PIT. Scale bar, 50 um.

FIG. 5B is a graph showing irradiation dose dependent and targetspecific cell death in response to Pan-1R700 (PanIR) mediated PIT. Dataare means±s.e.m. (n=at least 4, *** P<0.001 vs. non treatment control,Student's t test).

FIG. 5C is a digital image showing EGFR expressing cell specificnecrotic cell death was induced by Pan-1R700 mediated PIT. Scale bar, 50μm. DIC: differential interference contrast FIG. 6A is a digital imageshowing specific localization of panitumumab-IR700 conjugate (Pan-IR700)in a mouse previously administered A431 cells. HER1 positive A431 tumor(left dorsum) was selectively visualized as early as 1 d after Pan-1R700injection (50 μg). HER1 negative 3T3/HER2 tumor (right dorsum) did notshow detectable fluorescence (n=5 mice).

FIG. 6B is a graph showing the IR700 signal intensity in A431 tumorsover time following injection of two different doses (50 μg and 300 μg)of Pan-IR700. Data are means±s.e.m. (n=4 each mice).

FIG. 6C is a graph showing the tumor to background ratio of IR700fluorescence intensity in A431 tumors over time following injection oftwo different doses (50 μg and 300 μg) of Pan-IR700. Data aremeans±s.e.m. (n=4 each mice).

FIG. 6D is a digital image showing the biodistribution of Pan-1R700.A431 tumors (both sides of dorsum) were selectively visualized withIR700 fluorescence as early as 1 day after Pan-1R700 injection (300 μg).Right side of the tumor was irradiated with near infrared (NIR) light onday 1, while left side of the tumor was covered with black tape. Tumorshrinkage was confirmed on day 7. Dashed line: irradiated tumor, solidline: non-irradiated tumor.

FIG. 6E is a graph showing mean tumor volume following administration invivo of Pan-IR700 or carrier alone followed by PIT (30 J cm⁻²). PIT wasperformed on day 1 after Pan-IR700 injection (day 5 after tumorinoculation). Data are means±s.e.m. (at least n=12 mice in each group,*** P<0.001 vs. other control groups, Kruskal-Wallis test withpost-test).

FIG. 6F is a graph showing survival time following administration invivo of Pan-IR700 or carrier alone followed by PIT (30 J cm⁻²) (at leastn=12 mice in each group, *** P<0.001 vs. other control groups, log-ranktest with Bonferroni's correction for multiplicity.

FIG. 6G is a digital image showing hematoxylin and eosin stainedhistology images (×40 and ×200) 4 days after PIT treated (right) anduntreated (left) tumors. n=5 mice; Scale bar, 100 um. Pan: panitumumab.

FIG. 6H is a graph showing that high-dose administration of Pan-1R700lead to higher antitumor efficacy of Pan-1R700 mediated PIT for A431tumors in vivo. Tumor growth inhibition by Pan-1R700 mediated PIT wasPan-1R700 dose-dependently observed. Data are means±s.e.m. (at leastn=12 mice in each group).

FIG. 7 is a digital image showing the biodistribution of J591-1R700.PC3-PIP tumors were selectively visualized with IR700 fluorescence afterJ591-1R700 injection (100 μg). Right side of the tumor was irradiatedwith near infrared (NIR) light on days 4, 12, and 13 while left side ofthe tumor was covered with black tape.

Tumor shrinkage was confirmed on day 5.

FIG. 8 is a digital image showing the microscopic observation of beforeand after PIT for various cells in the presence of with Tra-1R700 forHER2+ cells, Pan-IR700 for HER1+ cells, and huJ591-1R700 for PSMA+cells. Scale bar, 50 μm. DIC: differential interference contrast.

FIG. 9A is a schematic drawing showing a schema for explaining selectivecancer therapy with PIT in the context of other physical cancertherapies employing electro-magnetic wave irradiation. Although otherphysical cancer therapies induce different types of damages in thenormal tissue, PIT dedicatedly damages cancer cells without damagingnormal cells or tissues.

FIG. 9B is a schematic drawing showing a schema for explainingphoto-physical, chemical and biological basis of PIT. Humanizedantibodies are employed as a delivery vehicle because of its highestbinding specificity, greatest in vivo target delivery, lowimmunogenecity among the clinically applicable targeting reagents. Ahydrophilic phtalocyanine is employed as an activatable cytotoxic“Nano-dynamite” reagent because of its great absorption of near infraredlight of 700 nm and strong cytotoxicity induced only when associatingwith the cell membrane. Near infrared light of 700 nm is employed as aninitiator for activating cytotoxicity because of its high energy amongnon-harmful non-ionizing photons and great in vivo tissue penetration.

FIGS. 10A-D. Samples of 1R700-conjugated Panitumumab (Pan-1R700) atconcentrations of 2.5, 5, 20, and 40 jag/mL were prepared by dilutingwith PBS. (A) Fluorescence intensity image of Pan-IR700 solution:Fluorescence intensities were decreased according to decrease ofconcentration of Pan-1R700. (B) Fluorescence lifetime (FLT) image ofPan-IR700 solution: The FLT at various concentrations of Pan-1R700solutions was almost the same value, 3.56+/−0.081 ns; 3.62 (2.5 pg/mL),3.58 (5 pg/mL), 3.44 (20 pg/mL), 3.60 ns (40 pg/mL). (C) LEDlight-irradiation for A431 cell pellets changes FLTs. A431 cell lineincubated with Pan-IR700 for 24 hours were treated with PIT at doses of0, 8, 15 and 30 J/cm². FLT shortened to 3.09, 2.94 and 2.85 ns, comparedwith 3.28 ns before light exposure. These represented shortenings of9.1, 10.1 and 13.1%, respectively. (D) FLT of A431 pellets depends onthe incubation time with Pan-IR700. FLT values escalate with incubationtime with Pan-IR700. FLT changes from 2.98 ns (1 hour) to 3.42 ns (24hours).

FIG. 11 is a digital image showing serial fluorescence (lower) anddifferential interference contrast (DIC) microscopic images (upper) ofA431 cells, which were pre-incubated with Pan-IR700 (10 μg/m0 at 37° C.for 24 h, 5, 15, 60 and 90 sec after start exposing NIR light. Pan-IR700gradually internalized into cytoplasm in A431 cells after bound to cellmembrane up to 24 h post-injection. Morphological changes on DIC becomeseverer by exposing more dose of NIR light. Scale bars, 50 μm.

FIGS. 12A-D. Comparison with FLTs of irradiated tumors (dark gray bar)and non-irradiated tumors (bright gray bar). (A) FLT images before andafter PIT at the dose of 10, 30, 50 J/cm² in the same mouse which wasinoculated with A431 cells on both sides of the mouse dorsum.Right-sided tumor was treated by PIT whereas the left was covered. FLTsof A431 tumors treated with PIT with 50 J/cm² (B), 30 J/cm² (C) and 10J/cm², (D) were plotted. PIT with the NIR light dose of 30 and 50 J/cm²demonstrated significant (P<0.05) shortenings in FLT immediately,compared with non-irradiated tumors. However, FLT did not significantlyshorten at a low dose of 10 J/cm². Transient prolongations of FLTs wereobserved at 6 hours after PIT likely due to uptake by reactivemacrophages. Mann-Whitney's U test was used for the statisticalanalysis.

FIGS. 13A-C. (A) FLT in PIT treated tumors with 50 and 30 J/cm²shortened significantly (p<0.01) compared with no treatment control (0J/cm²) (control). FLTs were immediately shortened to 69.1+/−10.9% and61.5+15.1% by PIT with 50 and 30 J/cm2, respectively. A431 tumorsirradiated with only 10 J/cm² showed no significant shortening of FLTimmediately after PIT. FLT shortened by only 7.7% at 48 hours after PITcompared with no treatment control. (B) FLT of non-irradiated tumors inPIT treated mice shortened slightly more than that in the untreated miceover time, but these changes were not significant. Student's t test wasused for the statistical analysis. (C) Histological specimens of A431tumors, which were treated with PIT at 0, 10, 30, and 50 J/cm², areshown. All specimens are stained with Hematoxylin and Eosin. Microscopicevaluation of treated tumors revealed various degrees of necrosis andmicro-hemorrhage with clusters of healthy or damaged tumor cells afterPIT. Necrotic damage was diffuse and intense and fewer surviving tumorcells are seen, when 30 and 50 J/cm² of NIR light was administered. Incontrast, when only 10 J/cm² of NIR light was administered, necroticcell damage was found in only limited areas within the tumor whilesubstantial amounts of healthy cancer foci remained. Scale indicates 50nm.

FIGS. 14A-C. A. The dynamic images of PEGylated Qdot800 after PIT. A431mice were injected with Pan-IR700, and 24 h later, NIR light (50 J/cm2)were irradiated to the right side tumor. Qdot800 was administered 1 hourafter PIT treatment. Only the right sided tumor was clearly shown within10 minutes. B. Time-signal intensity curves in the PIT-treated tumor(green; top), control tumor (blue; middle), and back (black; bottom). C.Fluorescence microscopy. IR700 signal shows the survived A431 cells.Qdot800 was broadly distributed in the PIT-treated tumor tissues andco-localization of IR700 and Qdot800 was partially observed, whereas,the signals of Qdot800 in control tumors were localized in the vicinityof main blood vessels.

FIGS. 15A-15B. A. The dynamic images of SPIO after PIT. A431 mice wereinjected with Pan-IR700, and 24 h later, NIR light (50 J/cm2) wereirradiated to the right side tumor. SPIO was administered 1 h after PITtreatment. Only the right-sided tumor was clearly shown up within 5 min.B. Prucian blue staining and HE staining.

FIGS. 16A-16D. A. The dynamic images of Pan-IR800 after PIT. A431 micewere injected with Pan-IR700, and 24 h later, NIR light (50 J/cm2) wereirradiated to the right side tumor. Pan-IR800 was administered 1 h afterPIT treatment. Only the right sided tumor was clearly shown up within 10min. B. Pan-IR800 can be quickly accumulated in the PIT-treated tumorsin dependent on irradiated light dose. Signal was not observed in thecontrol tumors. C and D. 24 h after PIT, Pan-IR800 cannot be taken up bythe tumor, likely because basement of blood vessels was repaired(intrestitaial pressure was recovered) or blood flow was stopped.

FIGS. 17A-F. A-C The dynamic images of daunorubicin containing liposomeafter PIT. A431 mice were injected with Pan-IR700, and 24 h later, NIRlight (50 J/cm2) were irradiated to the right side tumor. Daunorubicincontaining liposome was administered 1 h after PIT treatment. Only theright sided tumor was clearly shown up within 30 min. D. Fluorescencemicroscopic studies. IR700 signal shows the survived A431 cells.Daunorubicin containing liposome was broadly distributed in thePIT-treated tumor tissues and co-localization of IR700 and Qdot800 waspartially observed, whereas, the signals of Qdot800 in control tumorswere localized in the vicinity of main blood vessels. E. The combinationtherapy combined PIT with liposomes containing daunorubicinsignificantly suppressed tumor growth and (F) prolonged survival time ofA431 bearing mice.

FIG. 18 provides a schematic drawing showing an exemplary method fortreating tumors using PIT and chemotherapeutics, which can includeimaging of the tumor.

FIGS. 19A-19B. A. The dynamic images of Tra-IR800 after PIT. 3T3/HER2mice were injected with Tra-IR700, and 24 h later, NIR light (50 J/cm2)were irradiated to the right side tumor. Tra-IR800 was administered 1 hafter PIT treatment. Only the right sided tumor was clearly shown upwithin 10 min. White arrows show the site where light was insufficientlyirradiated to the 3T3HER2 tumors. Tra-IR800 can be accumulated in onlythe regions where the tumor was exposed to NIR light. B. The dynamicimages of Pan-IR800 after PIT. MDA-MB-468 bearing mice were injectedwith Pan-IR700, and 24 h later, NIR light (50 J/cm2) were irradiated tothe right side tumor. Pan-IR800 was administered 1 h after PITtreatment. Only the right sided tumor was clearly shown up within 10min.

FIGS. 20A-20C. A. The dynamic images of USPIO after PIT. A431 mice wereinjected with Pan-IR700, and 24 h later, NIR light (50 J/cm2) wereirradiated to the right side tumor. USPIO was administered 1 h after PITtreatment. Only the right sided tumor was clearly shown up within 5 min.B. Prucian blue staining and HE staining. C. The dynamic images of G6-Gdafter PIT. A431 mice were injected with Pan-IR700, and 24 h later, NIRlight (50 J/cm2) were irradiated to the right side tumor. G6-Gd wasadministered 1 h after PIT treatment. Only the right sided tumor wasclearly shown up within 5 min.

FIGS. 21A-21B. A. Fluorescence microscopic studies in the margin andcore regions of tumors. IR700 signal shows the survived A431 cells.Daunorubicin containing liposome was broadly distributed in thePIT-treated tumor tissues and co-localization of IR700 and Daunorubicincontaining liposome was partially observed. Especially in the coreregions, DX can be taken up in the locally necrotic regions. B. Thechange of body weight after therapy. There was no obvious differencebetween groups.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which a disclosed invention belongs. The singularterms “a,” “an,” and “the” include plural referents unless contextclearly indicates otherwise. Similarly, the word “or” is intended toinclude “and” unless the context clearly indicates otherwise.“Comprising” means “including.” Hence “comprising A or B” means“including A” or “including B” or “including A and B.”

Suitable methods and materials for the practice and/or testing ofembodiments of the disclosure are described below. Such methods andmaterials are illustrative only and are not intended to be limiting.Other methods and materials similar or equivalent to those describedherein can be used. For example, conventional methods well known in theart to which a disclosed invention pertains are described in variousgeneral and more specific references, including, for example, Sambrooket al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold SpringHarbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: ALaboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel etal., Current Protocols in Molecular Biology, Greene PublishingAssociates, 1992 (and Supplements to 2000); Ausubel et al., ShortProtocols in Molecular Biology: A Compendium of Methods from CurrentProtocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1999.

The sequences associated with all GenBank Accession numbers referencedherein are incorporated by reference for the sequence available on Jul.9, 2010.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Administration: To provide or give a subject an agent, such as anantibody-IR700 molecule, by any effective route. Exemplary routes ofadministration include, but are not limited to, topical, injection (suchas subcutaneous, intramuscular, intradermal, intraperitoneal,intratumoral, and intravenous), oral, ocular, sublingual, rectal,transdermal, intranasal, vaginal and inhalation routes.

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region which specifically recognizesand binds an epitope of an antigen, such as a tumor-specific protein.Antibodies are composed of a heavy and a light chain, each of which hasa variable region, termed the variable heavy (V_(H)) region and thevariable light (V_(L)) region. Together, the V_(H) region and the V_(L)region are responsible for binding the antigen recognized by theantibody.

Antibodies include intact immunoglobulins and the variants and portionsof antibodies well known in the art, such as Fab fragments, Fab′fragments, F(ab)′₂ fragments, single chain Fv proteins (“scFv”), anddisulfide stabilized Fv proteins (“dsFv”). A scFv protein is a fusionprotein in which a light chain variable region of an immunoglobulin anda heavy chain variable region of an immunoglobulin are bound by alinker, while in dsFvs, the chains have been mutated to introduce adisulfide bond to stabilize the association of the chains. The term alsoincludes genetically engineered forms such as chimeric antibodies (forexample, humanized murine antibodies), heteroconjugate antibodies (suchas, bispecific antibodies). See also, Pierce Catalog and Handbook,1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology,3^(rd) Ed., W. H. Freeman & Co., New York, 1997

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (κ). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs.” The extent of theframework region and CDRs have been defined (see, Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991, which is hereby incorporated byreference). The Kabat database is now maintained online. The sequencesof the framework regions of different light or heavy chains arerelatively conserved within a species, such as humans. The frameworkregion of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. Antibodies with different specificities (i.e.different combining sites for different antigens) have different CDRs.Although it is the CDRs that vary from antibody to antibody, only alimited number of amino acid positions within the CDRs are directlyinvolved in antigen binding. These positions within the CDRs are calledspecificity determining residues (SDRs).

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone of Blymphocytes or by a cell into which the light and heavy chain genes of asingle antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a murine antibody that specifically binds mesothelin.

A “humanized” immunoglobulin is an immunoglobulin including a humanframework region and one or more CDRs from a non-human (for example amouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulinproviding the CDRs is termed a “donor,” and the human immunoglobulinproviding the framework is termed an “acceptor.” In one embodiment, allthe CDRs are from the donor immunoglobulin in a humanizedimmunoglobulin. Constant regions need not be present, but if they are,they must be substantially identical to human immunoglobulin constantregions, i.e., at least about 85-90%, such as about 95% or moreidentical. Hence, all parts of a humanized immunoglobulin, exceptpossibly the CDRs, are substantially identical to corresponding parts ofnatural human immunoglobulin sequences. A “humanized antibody” is anantibody comprising a humanized light chain and a humanized heavy chainimmunoglobulin. A humanized antibody binds to the same antigen as thedonor antibody that provides the CDRs. The acceptor framework of ahumanized immunoglobulin or antibody may have a limited number ofsubstitutions by amino acids taken from the donor framework. Humanizedor other monoclonal antibodies can have additional conservative aminoacid substitutions which have substantially no effect on antigen bindingor other immunoglobulin functions. Humanized immunoglobulins can beconstructed by means of genetic engineering (see for example, U.S. Pat.No. 5,585,089).

A “human” antibody (also called a “fully human” antibody) is an antibodythat includes human framework regions and all of the CDRs from a humanimmunoglobulin. In one example, the framework and the CDRs are from thesame originating human heavy and/or light chain amino acid sequence.However, frameworks from one human antibody can be engineered to includeCDRs from a different human antibody. All parts of a humanimmunoglobulin are substantially identical to corresponding parts ofnatural human immunoglobulin sequences.

“Specifically binds” refers to the ability of individual antibodies tospecifically immunoreact with an antigen, such as a tumor-specificantigen, relative to binding to unrelated proteins, such as non-tumorproteins, for example β-actin. For example, a HER2-specific bindingagent binds substantially only the HER-2 protein in vitro or in vivo. Asused herein, the term “tumor-specific binding agent” includestumor-specific antibodies and other agents that bind substantially onlyto a tumor-specific protein in that preparation.

The binding is a non-random binding reaction between an antibodymolecule and an antigenic determinant of the T cell surface molecule.The desired binding specificity is typically determined from thereference point of the ability of the antibody to differentially bindthe T cell surface molecule and an unrelated antigen, and thereforedistinguish between two different antigens, particularly where the twoantigens have unique epitopes. An antibody that specifically binds to aparticular epitope is referred to as a “specific antibody”.

In some examples, an antibody (such as an antibody-IR700 molecule)specifically binds to a target (such as a cell surface protein) with abinding constant that is at least 10³ M⁻¹ greater, 10⁴ M⁻ greater or 10⁵M⁻¹ greater than a binding constant for other molecules in a sample orsubject. In some examples, an antibody (e.g., monoclonal antibody) orfragments thereof, has an equilibrium constant (Kd) of 1 nM or less. Forexample, an antibody binds to a target, such as tumor-specific proteinwith a binding affinity of at least about 0.1×10⁻⁸ M, at least about0.3×10⁻⁸ M, at least about 0.5×10⁻⁸ M, at least about 0.75×10⁻⁸ M, atleast about 1.0×10 M, at least about 1.3×10⁻⁸ M at least about 1.5×10⁻⁸M, or at least about 2.0×10 M. Kd values can, for example, be determinedby competitive ELISA (enzyme-linked immunosorbent assay) or using asurface-plasmon resonance device such as the Biacore T100, which isavailable from Biacore, Inc., Piscataway, N.J.

Antibody-IR700 molecule or antibody-IR700 conjugate: A molecule thatincludes both an antibody, such as a tumor-specific antibody, conjugatedto IR700. In some examples the antibody is a humanized antibody (such asa humanized monoclonal antibody) that specifically binds to a surfaceprotein on a cancer cell.

Antigen (Ag): A compound, composition, or substance that can stimulatethe production of antibodies or a T cell response in an animal,including compositions (such as one that includes a tumor-specificprotein) that are injected or absorbed into an animal. An antigen reactswith the products of specific humoral or cellular immunity, includingthose induced by heterologous antigens, such as the disclosed antigens.“Epitope” or “antigenic determinant” refers to the region of an antigento which B and/or T cells respond. In one embodiment, T cells respond tothe epitope, when the epitope is presented in conjunction with an MHCmolecule. Epitopes can be formed both from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of a protein.Epitopes formed from contiguous amino acids are typically retained onexposure to denaturing solvents whereas epitopes formed by tertiaryfolding are typically lost on treatment with denaturing solvents. Anepitope typically includes at least 3, and more usually, at least 5,about 9, or about 8-10 amino acids in a unique spatial conformation.Methods of determining spatial conformation of epitopes include, forexample, x-ray crystallography and nuclear magnetic resonance.

Examples of antigens include, but are not limited to, peptides, lipids,polysaccharides, and nucleic acids containing antigenic determinants,such as those recognized by an immune cell. In some examples, an antigenincludes a tumor-specific peptide (such as one found on the surface of acancer cell) or immunogenic fragment thereof.

Cancer: A malignant tumor characterized by abnormal or uncontrolled cellgrowth. Other features often associated with cancer include metastasis,interference with the normal functioning of neighboring cells, releaseof cytokines or other secretory products at abnormal levels andsuppression or aggravation of inflammatory or immunological response,invasion of surrounding or distant tissues or organs, such as lymphnodes, etc. “Metastatic disease” refers to cancer cells that have leftthe original tumor site and migrate to other parts of the body forexample via the bloodstream or lymph system. In one example, the cellkilled by the disclosed methods is a cancer cell.

Contacting: Placement in direct physical association, including both asolid and liquid form. Contacting can occur in vitro, for example, withisolated cells, such as tumor cells, or in vivo by administering to asubject (such as a subject with a tumor).

Decrease: To reduce the quality, amount, or strength of something. Inone example, a therapeutic composition that includes one or moreantibody-IR700 molecules decreases the viability of cells to which theantibody-IR700 molecule specifically binds, following irradiation of thecells with NIR (for example at a wavelength of about 680 nm) at a doseof at least 1 J cm²⁻, for example as compared to the response in theabsence of the antibody-IR700 molecule. In some examples such a decreaseis evidenced by the killing of the cells. In some examples, the decreasein the viability of cells is at least 20%, at least 50%, at least 75%,or even at least 90%, relative to the viability observed with acomposition that does not include an antibody-IR700 molecule. In otherexamples, decreases are expressed as a fold change, such as a decreasein the cell viability by at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 8-fold, at least 10-fold, or even atleast 15 or 20-fold, relative to the viability observed with acomposition that does not include an antibody-IR700 molecule. Suchdecreases can be measured using the methods disclosed herein.

IR700 (IRDye@ 700DX): A dye having the following formula:

Currently commercially available from LI-COR (Lincoln, Nebr.). IR700 hasseveral favorable chemical properties. Amino-reactive IR700 is arelatively hydrophilic dye and can be covalently conjugated with anantibody using the NHS ester of IR700. IR700 also has more than 5-foldhigher extinction coefficient (2.1×10 M⁻¹ cm⁻¹ at the absorption maximumof 689 nm), than conventional photosensitizers such asthehematoporphyrin derivative Photofrin® (1.2×10³ M⁻¹cm⁻¹ at 630 nm),meta-tetrahydroxyphenylchlorin; Foscan® (2.2×10⁴ M⁻¹cm⁻¹ at 652 nm), andmono-L-aspartylchlorin e6; NPe6/Laserphyrin® (4.0×10⁴ M⁻¹cm⁻¹ at 654nm).

Pharmaceutical composition: A chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect when properlyadministered to a subject. A pharmaceutical composition can include atherapeutic agent, such as one or more antibody-IR700 molecules. Atherapeutic or pharmaceutical agent is one that alone or together withan additional compound induces the desired response (such as inducing atherapeutic or prophylactic effect when administered to a subject). In aparticular example, a pharmaceutical composition includes atherapeutically effective amount of at least one antibody-IR700molecule.

Pharmaceutically acceptable vehicles: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 19th Edition (1995), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic compounds, such as one or more antibody-IR700 molecules.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Photoimmunotherapy (PIT): A molecular targeted therapeutic that utilizesa target-specific photosensitizer based on a near infrared (NIR)phthalocyanine dye, IR700, conjugated to monoclonal antibodies (MAb)targeting cell surface receptors. In one example the cell surfacereceptor is one found specifically on cancer cells, such as HER1, HER2or PSMA, and thus PIT can be used to kill such cells. Cell death of thecells occurs when the antibody-IR700 molecule binds to the cells and thecells are irradiated with NIR, while cells that do not express the cellsurface receptor recognized by the antibody-IR700 molecule are notkilled in significant numbers.

Subject or patient: A term that includes human and non-human mammals. Inone example, the subject is a human or veterinary subject, such as amouse. In some examples, the subject is a mammal (such as a human) whohas cancer, or is being treated for cancer.

Therapeutically effective amount: An amount of a composition that alone,or together with an additional therapeutic agent(s) (such as achemotherapeutic agent) sufficient to achieve a desired effect in asubject, or in a cell, being treated with the agent. The effectiveamount of the agent (such as an antibody-IR700 molecule) can bedependent on several factors, including, but not limited to the subjector cells being treated, the particular therapeutic agent, and the mannerof administration of the therapeutic composition. In one example, atherapeutically effective amount or concentration is one that issufficient to prevent advancement (such as metastasis), delayprogression, or to cause regression of a disease, or which is capable ofreducing symptoms caused by the disease, such as cancer. In one example,a therapeutically effective amount or concentration is one that issufficient to increase the survival time of a patient with a tumor.

In one example, a desired response is to reduce or inhibit one or moresymptoms associated with cancer. The one or more symptoms do not have tobe completely eliminated for the composition to be effective. Forexample, administration of a composition containing an antibody-IR700molecule followed by irradiation can decrease the size of a tumor (suchas the volume or weight of a tumor, or metastasis of a tumor), forexample by at least 20%, at least 50%, at least 80%, at least 90%, atleast 95%, at least 98%, or even at least 100%, as compared to the tumorsize in the absence of the antibody-IR700 molecule. In one particularexample, a desired response is to kill a population of cells by adesired amount, for example by killing at least 20%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98%, or even at least 100% of the cells, as compared to the cellkilling in the absence of the antibody-IR700 molecule and irradiation.In one particular example, a desired response is to increase thesurvival time of a patient with a tumor (or who has had a tumor recentlyremoved) by a desired amount, for example increase survival by at least20%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 98%, or even at least 100%, as compared tothe survival time in the absence of the antibody-IR700 molecule andirradiation.

The effective amount of an agent that includes one of the disclosedantibody-IR700 molecules, that is administered to a human or veterinarysubject will vary depending upon a number of factors associated withthat subject, for example the overall health of the subject. Aneffective amount of an agent can be determined by varying the dosage ofthe product and measuring the resulting therapeutic response, such asthe regression of a tumor. Effective amounts also can be determinedthrough various in vitro, in vivo or in situ immunoassays. The disclosedagents can be administered in a single dose, or in several doses, asneeded to obtain the desired response. However, the effective amount ofan agent can be dependent on the source applied, the subject beingtreated, the severity and type of the condition being treated, and themanner of administration.

In particular examples, a therapeutically effective dose of anantibody-IR700 molecule is at least 0.5 milligram per 60 kilogram(mg/kg), at least 5 mg/60 kg, at least 10 mg/60 kg, at least 20 mg/60kg, at least 30 mg/60 kg, at least 50 mg/60 kg, for example 0.5 to 50mg/60 kg, such as a dose of 1 mg/60 kg, 2 mg/60 kg, 5 mg/60 kg, 20 mg/60kg, or 50 mg/60 kg, for example when administered iv. In anotherexample, a therapeutically effective dose of an antibody-IR700 moleculeis at least 10 μg/kg, such as at least 100 μg/kg, at least 500 μg/kg, orat least 500 μg/kg, for example 10 μg/kg to 1000 μg/kg, such as a doseof 100 μg/kg, 250 μg/kg, about 500 μg/kg, 750 μg/kg, or 1000 μg/kg, forexample when administered intratumorally or ip. In one example, atherapeutically effective dose is at least 1 μg/ml, such as at least 500μg/ml, such as between 20 μg/ml to 100 μg/ml, such as 10 μg/ml, 20μg/ml, 30 μg/ml, 40 μg/ml, 50 μg/ml, 60 μg/ml, 70 μg/ml, 80 μg/ml, 90μg/ml or 100 μg/ml administered in topical solution. However, oneskilled in the art will recognize that higher or lower dosages alsocould be used, for example depending on the particular antibody-IR700molecule. In particular examples, such daily dosages are administered inone or more divided doses (such as 2, 3, or 4 doses) or in a singleformulation. The disclosed antibody-IR700 molecules can be administeredalone, in the presence of a pharmaceutically acceptable carrier, in thepresence of other therapeutic agents (such as other anti-neoplasticagents).

Generally a suitable dose of irradiation following administration of theantibody-IR700 is at least 1 J cm⁻² at a wavelength of 660-740 nm, forexample, at least 10 J cm⁻² at a wavelength of 660-740 nm, at least 50 Jcm⁻² at a wavelength of 660-740 nm, or at least 100 J cm⁻² at awavelength of 660-740 nm, for example 1 to 500 J cm⁻² at a wavelength of660-740 nm. In some examples the wavelength is 660-710 nm. In specificexamples, a suitable dose of irradiation following administration of theantibody-IR700 molecule is at least 1.0 J cm⁻² at a wavelength of 680 nmfor example, at least 10 J cm⁻² at a wavelength of 680 nm, at least 50 Jcm⁻² at a wavelength of 680 nm, or at least 100 J cm⁻² at a wavelengthof 680 nm, for example 1 to 500 1.0 J cm⁻² at a wavelength of 680 nm. Inparticular examples, multiple irradiations are performed (such as atleast 2, at least 3, or at least 4 irradiations, such as 2, 3, 4, 5, 6,7, 8, 9 or 10 separate administrations), following administration of theantibody-IR700 molecule.

Treating: A term when used to refer to the treatment of a cell or tissuewith a therapeutic agent, includes contacting or incubating an agent(such as an antibody-IR700 molecule) with the cell or tissue. A treatedcell is a cell that has been contacted with a desired composition in anamount and under conditions sufficient for the desired response. In oneexample, a treated cell is a cell that has been exposed to anantibody-IR700 molecule under conditions sufficient for the antibody tobind to a surface protein on the cell, followed by irradiation, untilsufficient cell killing is achieved.

Tumor, neoplasia, malignancy or cancer: A neoplasm is an abnormal growthof tissue or cells which results from excessive cell division.Neoplastic growth can produce a tumor. The amount of a tumor in anindividual is the “tumor burden” which can be measured as the number,volume, or weight of the tumor. A tumor that does not metastasize isreferred to as “benign.” A tumor that invades the surrounding tissueand/or can metastasize is referred to as “malignant.” A “non-canceroustissue” is a tissue from the same organ wherein the malignant neoplasmformed, but does not have the characteristic pathology of the neoplasm.Generally, noncancerous tissue appears histologically normal. A “normaltissue” is tissue from an organ, wherein the organ is not affected bycancer or another disease or disorder of that organ. A “cancer-free”subject has not been diagnosed with a cancer of that organ and does nothave detectable cancer.

Exemplary tumors, such as cancers, that can be treated with the claimedmethods include solid tumors, such as breast carcinomas (e.g. lobularand duct carcinomas), sarcomas, carcinomas of the lung (e.g., non-smallcell carcinoma, large cell carcinoma, squamous carcinoma, andadenocarcinoma), mesothelioma of the lung, colorectal adenocarcinoma,stomach carcinoma, prostatic adenocarcinoma, ovarian carcinoma (such asserous cystadenocarcinoma and mucinous cystadenocarcinoma), ovarian germcell tumors, testicular carcinomas and germ cell tumors, pancreaticadenocarcinoma, biliary adenocarcinoma, hepatocellular carcinoma,bladder carcinoma (including, for instance, transitional cell carcinoma,adenocarcinoma, and squamous carcinoma), renal cell adenocarcinoma,endometrial carcinomas (including, e.g., adenocarcinomas and mixedMullerian tumors (carcinosarcomas)), carcinomas of the endocervix,ectocervix, and vagina (such as adenocarcinoma and squamous carcinoma ofeach of same), tumors of the skin (e.g., squamous cell carcinoma, basalcell carcinoma, malignant melanoma, skin appendage tumors, Kaposisarcoma, cutaneous lymphoma, skin adnexal tumors and various types ofsarcomas and Merkel cell carcinoma), esophageal carcinoma, carcinomas ofthe nasopharynx and oropharynx (including squamous carcinoma andadenocarcinomas of same), salivary gland carcinomas, brain and centralnervous system tumors (including, for example, tumors of glial,neuronal, and meningeal origin), tumors of peripheral nerve, soft tissuesarcomas and sarcomas of bone and cartilage, and lymphatic tumors(including B-cell and T-cell malignant lymphoma). In one example, thetumor is an adenocarcinoma.

The methods can also be used to treat liquid tumors, such as alymphatic, white blood cell, or other type of leukemia. In a specificexample, the tumor treated is a tumor of the blood, such as a leukemia(for example acute lymphoblastic leukemia (ALL), chronic lymphocyticleukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenousleukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocyticleukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cellleukemia), lymphomas (such as Hodgkin's lymphoma and non-Hodgkin'slymphoma), and myelomas).

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits the desired activity. In one example, “underconditions sufficient for” includes administering an antibody-IR700molecule to a subject sufficient to allow the antibody-IR700 molecule tobind to cell surface proteins. In particular examples, the desiredactivity is killing the cells to which the antibody-IR700 molecule isbound, following therapeutic irradiation of the cells.

Untreated cell: A cell that has not been contacted with a desired agent,such as an antibody-IR700 molecule. In an example, an untreated cell isa cell that receives the vehicle in which the desired agent wasdelivered.

Disclosure of certain specific examples is not meant to exclude otherembodiments. In addition, any treatments described herein are notnecessarily exclusive of other treatment, but can be combined with otherbioactive agents or treatment modalities.

Overview of Technology

Conventional photodynamic therapy (PDT) for cancer therapy is based onthe preferential accumulation of a photosensitizer in tumor to producephototoxicity with minimal damage to surrounding tissue (Dougherty etal. J Natl Cancer Inst 90:889-905, 1998). Traditionally, PDT is thoughtto be mediated by the generation of ROS, especially singlet oxygen, inthe presence of oxygen (Dougherty et al. J Natl Cancer Inst 90:889-905,1998). However, to the extent that existing photosensitizers lack tumorselectivity, considerable damage can be seen in normal tissues leadingto dose limiting toxicity. Thus, current methods of PDT would beimproved if more selective targeting of the photosensitizer and moreefficient phototoxicity per photon absorbed was possible.

Disclosed herein are highly targeted photosensitizers, referred to asantibody-IR700 molecules. The photosensitizer, IR700, is excited in theNIR range leading to deeper tissue penetration resulting in successfuleradication of subcutaneously xenografted tumors after only a singledose of external NIR light irradiation. Targeted phototoxicity appearsto be primarily dependent on binding of the antibody-1R700 molecules tothe cell membrane and to a lesser extent on internalization and ROSformation. The fluorescence induced by the conjugate can be used tonon-invasively guide both PIT and monitor the results of therapy.

Although a targeted photosensitizer can distribute throughout the body,it is only active where intense light is applied, reducing thelikelihood of off-target effects. In contrast, existing photosensitizersare poorly selective small molecules which bind not only to cancer cellsbut also to normal cells, including the skin and other epithelialsurfaces, resulting in unwanted phototoxicity. In addition, targetspecific delivery of conventional photosensitizers is theoreticallydifficult because, after reaching the cell, the agent must still beinternalized into organelles, such as mitochondria, to be mosteffective. Various combinations of conventional photosensitizers andMAbs have been tested to improve selectivity (Mew et al., J Immunol130:1473-1477, 1983; Sobolev et al., Prog Biophys Mol Biol 73:51-90,2000; Carcenac et al., Br J Cancer 85:1787-93, 2001; Vrouenraets et al.,Cancer Res 59:1505-13, 1999; Vrouenraets et al., Cancer Res61:1970-1975, 2001; Hamblin et al., Cancer Res 56:5205-10, 1996; Mew etal., Cancer Res 45:4380-6, 1985). However, these have had limitedsuccess especially when measured by in vivo therapeutic effects, forexample because conventional photosensitizers have low extinctioncoefficients that require conjugation of large numbers ofphotosensitizers to a single antibody molecule thus, potentiallydecreasing binding affinity, because conventional photosensitizers aremostly hydrophobic leading to difficulties in conjugatingphotosensitizers to antibodies without compromising the immunoreactivityand in vivo target accumulation, and because conventionalphotosensitizers generally absorb light in the visible range reducingtissue penetration.

It is shown herein that antibody-based photosensitizers (such asmAb-based photosensitizers), which are activated by NIR light fortargeted photoimmunotherapy (PIT) only when bound to the target moleculeon the cancer cellular membrane. The fluorophore IR700 (Licor Co.Lincoln, Nebr.) can become a photosensitizer when conjugated to anantibody specific for a cell surface receptor and can thus be used fortarget specific photodynamic therapy of undesired cells, such as tumoror cancer cells. Further, because these agents also emit a diagnosticfluorescence, they can be used to direct the application of light tominimize light exposure to non-relevant tissues and non-invasivelymonitor therapeutic effects. Based on the similarity of thephototoxicity induced with three different MAbs against severaldifferent cells expressing various numbers of respective targetmolecules and considering the potentially additive benefits fromimmunotherapy this method can be generally applicable to other mAbs(such as those disclosed in Nanus et al., J. Urology 170:S84-S88, 2003and van Dongen et al., Adv Drug Deliv Rev 56:31-52, 2004).

When IR700 was conjugated with an anti-EGFR antibody (HER1 or HER2) or aPSMA antibody, cells that selectively bound the conjugate were killedupon exposure to 680 nm near-infrared (NIR) light. Based on this novelobservation patient therapies are provided. Since thisantibody-dependent target-cell specific photodynamic therapy is achievedwith NIR light (e.g., 680 nm) excitation and showed highly selectivelycytotoxic effects only upon antibody binding, this newantibody-dependent target-cell specific photodynamic therapy using IR700can be used in cancer patients as a way to personalize cancer therapywith minimal side effects.

The selectivity of the antibody-IR700 conjugate is derived from itsactivation after binding to the cell membrane of target cells; unboundconjugate does not contribute to phototoxicity. Short term viabilityassays, as well as long term proliferation assays, demonstrated that theconjugate was capable of inducing specific cell death. When co-culturesof receptor-positive and -negative cells were treated, only thereceptor-positive cells were killed despite the presence of unboundantibody—1R700 in the culture medium. This selective cell killingminimizes damage to normal cells.

The antibody-1R700 molecule must be bound to the cellular membrane to beactive. For instance, the rupture of endolysosome occurred within asecond of light exposure. Cell death induced by singlet oxygen generallyinduces a slower apoptotic cell death. Since cell membrane damage was soquickly induced even at 4° C. by this method, it is hypothesized thatcell death is caused by the rapid expansion of locally heated water withrelatively minor effects due to singlet oxygen effects.

Treatment with sodium azide, a redox and singlet oxygen scavenger, onlypartially reduced the phototoxicity but did not totally eliminate theeffectiveness of the conjugate. This indicates that ROS generation is aminor part of the phototoxic effect. The observation that phototoxicitywas induced after incubation with antibody-1R700 after only 1 hour at 4°C. indicates that internalization of the conjugate is not required foractivity. This differs from current PDT agents that requireintracellular localization to be effective. Video microscopydemonstrated rapid visible damage to the membrane and lysosomes afterexposure to light, following incubation for more than 6 hours at 37° C.,when the antibody-conjugate was internalized.

The disclosed antibody-1R700 conjugates permitted detection of targetedtissue. This can permit specific lesions to be identified with PITrather than irradiating the entire field. Doses required for diagnosis(50 μg) were significantly lower than those required for therapy (300μg). Improved intratumoral distribution of antibody occurred with thetherapeutic dose. Because both bound and unbound agent fluoresces, thereis relatively high background signal at therapeutic doses. Nevertheless,after PIT, the fluorescence of the treated tumors decreased andeventually disappeared, providing a means of monitoring the treatment

Free IR700 and catabolized IR700, are readily excreted into urine within1 hour without accumulation in any specific organ. The other componentof PIT, light irradiation with NIR (e.g., at 690 nm) is unlikely to betoxic except at thermal doses. There should be no limitations on thecumulative irradiation dose of NIR light, unlike ionizing radiation suchas x-ray or gamma-ray (FIG. 9). Therefore, repeated PIT can be used forlong term management of cancer patients. It was observed that repeatedPIT with 3 different regimens (3 or 4 fractionated NIR irradiations at asingle dose of antibody-IR700 conjugate and 4 cycles of PIT every 2weeks after multiple doses of antibody) controlled tumor regrowth,resulting in tumor free survival of more than 4 months.

It is also shown herein that use of antibody-1R700 conjugates with PITenhances delivery of nano-sized agents to the tumor, for example forabout 8 hours following PIT. Nano-sized agents can target vascularendothelial cells by activating with humeral factor including depletionof vascular endothelial growth factor (VEGF) or damaging cells usingvascular toxic agents. Based on these observations, methods are providedfor enhancing delivery of other anti-neoplastic therapies to the tumorfollowing PIT. In one example, delivery of the anti-neoplastic therapyto the tumor (or effectiveness of the anti-neoplastic therapy) isincreased by at least 20/%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 75%, at least 80%, at least 90%, or even at least95%, for example as compared to delivery of the anti-neoplastic therapyto the tumor (or effectiveness of the anti-neoplastic therapy) in theabsence of administration of the antibody-1R700 conjugates and PIT.

Methods for Killing Cells and Treating Tumors

The present disclosure provides methods for killing a cell, such as atarget cell. The cell expresses a protein on its surface, such as atumor specific antigen, that can specifically bind to an antibody thatis conjugated to the dye IR700 (referred to herein as an antibody-IR700molecule). The cell is contacted with a therapeutically effective amountof one or more antibody-IR700 molecules (for example in the presence ofa pharmaceutically acceptable carrier, such as a pharmaceutically andphysiologically acceptable fluid), under conditions that permit theantibody to specifically bind to the cell surface protein. For example,the antibody-IR700 molecule can be present in a pharmaceuticallyeffective carrier, such as water, physiological saline, balanced saltsolutions (such as PBS/EDTA), aqueous dextrose, sesame oil, glycerol,ethanol, combinations thereof, or the like, as a vehicle. The carrierand composition can be sterile, and the formulation suits the mode ofadministration.

After contacting or administering the one or more antibody-IR700molecules under conditions that allow the one or more antibody-IR700molecules to bind to their target on a cell surface, the cell is thenirradiated under conditions that permit killing of the cells, forexample irradiation at a wavelength of 660 to 710 nm at a dose of atleast 1 J cm⁻². In one example, there is at least 10 minutes, at least30 minutes, at least 1 hour, at least 4 hours, at least 8 hours, atleast 12 hours, or at least 24 hours (such as 1 to 4 hours, 30 minutesto 1 hour, 10 minutes to 60 minutes, or 30 minutes to 8 hours) inbetween contacting the cell with the antibody-IR700 molecules and theirradiation. The NIR excitation light wavelength allows penetration ofat least several centimeters into tissues. For example, by usingfiber-coupled laser diodes with diffuser tips, NIR light can bedelivered within several centimeters of otherwise inaccessible tumorslocated deep to the body surface. In addition to treating solid cancers,circulating tumor cells can be targeted since they can be excited whenthey traverse superficial vessels (for example using the NIR LEDwearable devices disclosed herein). The disclosed methods can also beused as a therapy for transplant rejection.

The method also includes contacting the cell with one or more additionaltherapeutic agents. The inventors have determined that there is about an8 hour window following irradiation (for example irradiation at awavelength of 660 to 710 nm at a dose of at least 10 J cm⁻², at least 20J cm⁻², at least 30 J cm⁻², at least 40 J cm⁻², at least 50 J cm⁻², atleast 70 J cm⁻², at least 80 J cm⁻² or at least 100 J cm⁻², such as atleast 10 to 100 J cm⁻²), during which uptake of additional agents (e.g.,nano-sized agents, such as those about at least 1 nm in diameter, atleast 10 nm in diameter, at least 100 nm in diameter, or at least 200 nmin diameter, such as 1 to 500 nm in diameter) by the PIT-treated cellsis unexpectedly and superiorly enhanced. Thus, one or more additionaltherapeutic agents can be contacted with the cell contemporaneously orsequentially with the PIT. In one example, the additional therapeuticagents are administered after the irradiation, for example, about 0 to 8hours after irradiating the cell (such as at least 10 minutes, at least30 minutes, at least 60 minutes, at least 2 hours, at least 3 hours, atleast 4, hours, at least 5 hours, at least 6 hours, or at least 7 hoursafter the irradiation, for example no more than 10 hours, no more than 9hours, or no more than 8 hours, such as 1 hour to 10 hours, 1 hour to 9hours 1 hour to 8 hours, 2 hours to 8 hours, or 4 hours to 8 hours afterirradiation). In another example, the additional therapeutic agents areadministered just before the irradiation (such as about 10 minutes to120 minutes before irradiation, such as 10 minutes to 60 minutes or 10minutes to 30 minutes before irradiation).

In some examples, combining the antibody-IR700 molecules/PIT with theadditional therapy (such as anti-neoplastic agents), enhances theeffectiveness of the treatment of the tumor. For example, combining theantibody-IR700 molecules/PIT with the additional therapy (such asanti-neoplastic agents) can result in a tumor volume that is less thanthe tumor volume would be if it were treated with either theantibody-IR700 molecules/PIT alone or the additional therapy alone, thatis, there is a synergistic effect. In one example, the volume of a tumortreated with the combination therapy is at least 2-fold, at least3-fold, at least 4-fold, or even at least 5-fold smaller than the volumeof a tumor treated with either the antibody-IR700 molecules/PIT alone orthe additional therapy alone (for example after at least 7 days, atleast 10 days, at least 14 days, at least 30 days, at least 60 days, atleast 90 days, or at least 120 days after the treatment). In oneexample, the volume of a tumor treated with the combination therapy isat least 5-fold, at least 6-fold, at least 7-fold, or even at least10-fold smaller than the volume of a control untreated tumor (forexample after at least 7 days, at least 10 days, at least 14 days, atleast 30 days, at least 60 days, at least 90 days, or at least 120 daysafter the treatment). In another or additional example, combining theantibody-IR700 molecules/PIT with the additional therapy (such asanti-neoplastic agents) can increase the survival time of a subjecthaving a tumor relative to the survival time of the subject if the tumorwas treated with either the antibody-IR700 molecules/PIT alone or theadditional therapy alone, that is, there is a synergistic effect. In oneexample, the survival time of a subject having a tumor treated with thecombination therapy is at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 6-fold, at least 7-fold, or at least10-fold longer than survival time of a subject having a tumor treatedwith either the antibody-IR700 molecules/PIT alone or the additionaltherapy alone (for example after a specified period of time, such as atleast 14 days, at least 30 days, at least 60 days, at least 90 days, atleast 120 days, at least 6 months, at least 12 months, at least 24months, or at least 5 years after the treatment, more subjects treatedwith the combination therapy will be alive than if treated with eithertherapy alone). In one example, the survival time of a subject having atumor treated with the combination therapy is at least 5-fold, at least10-fold, at least 15-fold, or even at least 20-fold greater than thesurvival time of a subject having an untreated tumor (for example afterat least 7 days, at least 10 days, at least 14 days, at least 30 days,at least 60 days, at least 90 days, at least 120 days after thetreatment at least 6 months, at least 12 months, at least 24 months, orat least 5 years after the treatment, more subjects treated with thecombination therapy will be alive than if untreated).

Exemplary additional therapeutic agents include anti-neoplastic agents,such as chemotherapeutic and anti-angiogenic agents or therapies, suchas radiation therapy. In one example the agent is a chemotherapyimmunosuppressant (such as Rituximab, steroids) or a cytokine (such asGM-CSF). Chemotherapeutic agents are known in the art (see for example,Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison'sPrinciples of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed., 2000Churchill Livingstone, Inc; Baltzer and Berkery. (eds): Oncology PocketGuide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; FischerKnobf, and Durivage (eds): The Cancer Chemotherapy Handbook, 4th ed. St.Louis, Mosby-Year Book, 1993). Exemplary chemotherapeutic agents thatcan be used with the methods provided herein include but are not limitedto, carboplatin, cisplatin, paclitaxel, docetaxel, doxorubicin,epirubicin, topotecan, irinotecan, gemcitabine, iazofurine, gemcitabine,etoposide, vinorelbine, tamoxifen, valspodar, cyclophosphamide,methotrexate, fluorouracil, mitoxantrone, Doxil (liposome encapculateddoxiorubicine) and vinorelbine. In some examples, the additionaltherapeutic agent is conjugated to (or otherwise associated with) ananoparticle, such as one at least 1 nm in diameter (for example atleast 10 nm in diameter, at least 30 nm in diameter, at least 100 nm indiameter, at least 200 nm in diameter, at least 300 nm in diameter, atleast 500 nm in diameter, or at least 750 nm in diameter, such as 1 nmto 500 nm, 1 nm to 300 nm, 1 nm to 100 nm, 10 nm to 500 nm, or 10 nm to300 nm in diameter).

The methods can be used to kill cells in vitro, for example byincubating the cells with the antibody-IR700 molecules and one or moretherapeutic agents in culture, or in vivo, for example, by administeringone or more antibody-IR700 molecules and one or more therapeutic agentsto the subject. For example, a subject to be treated can be administereda therapeutically effective amount of one or more antibody-IR700molecules, followed by irradiating the subject (or a tumor or tumor cellin the subject) with a therapeutic dose of irradiation andadministration of one or more additional therapeutic agents (such aswithin about 8 hours of the irradiation).

In one example, contacting target cells with one or more antibody-IR700molecules followed by irradiation and administration of an additionaltherapeutic agent kills the target cells that express a cell surfaceprotein that specifically binds to the antibody. For example, thedisclosed methods can kill at least 10%, for example at least 20%, atleast 40%, at least 50%, at least 80%, at least 90%, or more of thetreated cells relative to the absence of treatment with one or moreantibody-IR700 molecules followed by irradiation and administration ofone or more therapeutic agents.

In one example, administration of one or more antibody-IR700 moleculesto a subject having a tumor, in combination with irradiation andadministration of one or more therapeutic agents, kills the cells thatexpress a cell surface protein that can specifically bind to theantibody, thereby treating the tumor. For example, the disclosed methodscan decrease the size or volume of a tumor, slow the growth of a tumor,decrease or slow metastasis of the tumor (for example by reducing thenumber of metastases or decreasing the volume or size of a metastasis),or combinations thereof. For example, the disclosed methods can reducetumor cell size or volume and/or a metastatic tumor cell volume (ornumber of metastatic tumors), such as by at least 10%, for example by atleast 20%, at least 40%, at least 50%, at least 80%, at least 90%, ormore, relative to the absence of administration of one or moreantibody-IR700 molecules followed by irradiation. In addition, thedisclosed methods can result in a decrease in the symptoms associatedwith a tumor and/or a metastatic tumor. In one example, administrationof the disclosed compositions slows the growth of a tumor, such as by atleast 10%, for example by at least 20%, at least 40%, at least 50%, atleast 80%, at least 90%, or more, relative to the absence ofadministration of the antibody-IR700 molecules followed by irradiation.Methods of monitoring tumor volume/size/metastasis are routine in theart. In some examples, the disclosed methods can increase a subject's(such as a subject with a tumor or who has had a tumor previouslyremoved) survival time, for example relative the absence ofadministration of one or more antibody-IR700 molecules, irradiation, andadministration of one or more therapeutic agents, such as an increase ofat least 20%, at least 40%, at least 50%, at least 80%, at least 90%, ormore. For example, the disclosed methods can increase a subject'ssurvival time by at least 3 months, at least 6 months, at least 12months, at least 18 months, at least 24 months, at least 36 months ormore, relative to average survival time in the absence of administrationof an antibody-IR700 molecule, irradiation, and administration of one ormore therapeutic agents.

Administration of therapeutically effective amounts of antibody-IR700molecules followed by therapeutically effective doses of irradiation andadministration of one or more therapeutic agents are capable ofselectively killing tumor cells in vivo, and are capable of decreasingthe weight or volume of a tumor in vivo. By selective killing of tumorcells relative to normal cells is meant that the methods are capable ofkilling tumor cells more effectively than normal cells such as, forexample, cells not expressing the cell surface protein that specificallybinds to the antibody administered.

The disclosed methods can be used to treat fixed tumors in the body aswell as tumors in the circulation (e.g., leukemia cells, metastases,circulating tumor cells). However, circulating cells, by their nature,cannot be exposed to light for very long. Thus, if the cell to be killedis one that is circulating throughout the body, the methods can beaccomplished by using a device that can be worn, or that covers parts ofthe body. For example, such a device can be worn for extended timeperiods. Everyday wearable items (e.g., wristwatches, jewelry (such as anecklace or bracelet), blankets, clothing (e.g., underwear, socks, andshoe inserts) and other everyday wearable items) which incorporate NIRemitting light emitting diodes (LEDs) and a battery pack, can be used.Such devices produce light on the skin underlying the device over longperiods leading to continual exposure of light to superficial vesselsover prolonged periods. Circulating tumor cells are exposed to the lightas they transit thru the area underlying the device. As an example, awristwatch or bracelet version of this device can include a series ofNIR LEDs with battery power pack to be worn for most of the day.

After administration of the one or more antibody-IR700 molecules (e.g.,intravenously), circulating cells bind the antibody-IR700 conjugate andbecome susceptible to killing by PIT. As these cells flow within thevessels adjacent to the LED present in the everyday wearable item (e.g.,bracelet or wristwatch), they would be exposed to NIR light renderingthem susceptible to cell killing. The dose of light may be adjustableaccording to diagnosis and cell type.

In some examples, the method further includes monitoring the therapy,such as killing of tumor cells. In such examples, the antibody-IR700conjugate is contacted with the cells and the cells irradiated asdescribed above. However, a lower dose of the antibody-IR700 conjugateand NIR light can be used (as cell killing may not be required, justmonitoring of the therapy). In one example, the amount of antibody-IR700conjugate administered for monitoring is at least 2-fold less (such asat least 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold less than thetherapeutic dose). In one example, the amount of antibody-IR700conjugate administered for monitoring is at least 20% or at least 25%less than the therapeutic dose. In one example, the amount of NIR lightused for monitoring is at least 1/1000 or at least 1/10,000 of thetherapeutic dose. This permits detection of the cells being treated. Forexample, by using such methods, the size of the tumor and metastases canbe monitored.

In some examples, the method is useful during surgery, such asendoscopic procedures. For example, after the antibody-IR700 conjugateis contacted with the cells and the cells irradiated as described above,this not only results in cell killing, but permits a surgeon or othermedical care provider to visualize the margins of a tumor, and helpensure that resection of the tumor (such as a tumor of the skin, breast,lung, colon, or prostate) is complete and that the margins are clear. Insome examples, a lower dose of the antibody-IR700 conjugate can be usedfor visualization, such as at least 2-fold less (such as at least 3-,4-, 5-, 6-, 7-, 8-, 9-, or 10-fold less than the therapeutic dose).

Methods are provided that permit detection or monitoring of cell killingin real-time. Such methods are useful for example, to ensure sufficientamounts ofantibody-IR700 molecules and/or one or more therapeuticagents, or sufficient amounts of irradiation, were delivered to the cellor tumor promote cell killing. These methods permit detection of cellkilling before morphological changes become evident. In one example, themethods include contacting a cell having a cell surface protein with atherapeutically effective amount of one or more antibody-IR700 molecules(such as at least 0.01 nM, at least 0.1 nM, at least 1 nM, or at least10 nM, such as 0.1 to 2 nM, 0.5 to 1.5 nM, such as 1 nM of the of one ormore antibody-IR700 molecules), wherein the antibody specifically bindsto the cell surface protein; irradiating the cell at a wavelength of 660to 740 nm and at a dose of at least 20 J cm⁻²; and detecting the cellwith fluorescence lifetime imaging about 0 to 48 hours after irradiatingthe cell (such as at least 1 hour, at least 2 hours, at least 4 hours,at least 6 hours, at least 12 hours, at least 18 hours, at least 24hours, at least 36 hours, at least 48 hours, or at least 72 hours afterirradiating the cell, for example 1 minute to 30 minutes, 10 minutes to30 minutes, 10 minutes to 1 hour, 1 hour to 8 hours, 6 hours to 24hours, or 6 hours to 48 hours after irradiating the cell), therebydetecting the cell killing in real-time. Shortening FLT serves as anindicator of acute membrane damage induced by PIT. Thus, the cell isirradiated under conditions sufficient to shorten IR700 FLT by at least25%, such as at least 40%, at least 50%, at least 60% or at least 75%.In one example, the cell is irradiated at a wavelength of 660 nm to 740nm (such as 680 nm to 700 nm) and at a dose of at least 20 J cm⁻² or atleast 30 J cm⁻², such as at least 40 J cm⁻² or at least 50⁻² J cm⁻² orat least 60 J cm⁻², such as 30 to 50 J cm⁻².

In some examples, methods of detecting cell killing in real timeincludes contacting the cell with one or more additional therapeuticagents, for example about 0 to 8 hours after irradiating the cell. Thereal-time imaging can occur before or after contacting the cell with oneor more additional therapeutic agents. For example, if insufficient cellkilling occurs after administration of the one or more antibody-IR700molecules as determined by the real-timing imaging, then the cell can becontacted with one or more additional therapeutic agents. However, insome examples, the cell is contacted with the antibody-IR700 moleculesand additional therapeutic agents prior to detecting the cell killing inreal-time.

Exemplary Cells

The target cell can be a cell that is not desired or whose growth is notdesired, such as a tumor cell. The cells can be growing in culture, orpresent in a mammal to be treated, such as a patient with cancer. Anytarget cell can be treated with the claimed methods. In one example, thetarget cell expresses a cell surface protein that is not substantiallyfound on the surface of other normal (desired) cells, an antibody can beselected that specifically binds to such protein, and an antibody-IR700molecule generated for that protein. In one example, the cell surfaceprotein is a tumor-specific protein. In one example, the cell surfaceprotein is CD25, which can be used to target cells associated withundesired transplant rejection.

In one example, the tumor cell is a cancer cell, such as a cell in apatient with cancer. Exemplary cells that can be killed with thedisclosed methods include cells of the following tumors: a liquid tumorsuch as a leukemia, including acute leukemia (such as acute lymphocyticleukemia, acute myelocytic leukemia, and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia and chronic lymphocyticleukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin'slymphoma, multiple myeloma, Waldenstrdm's macroglobulinemia, heavy chaindisease). In another example the cell is a solid tumor cell, such assarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostatecancer, hepatocellular carcinomna, lung cancer, colorectal cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (forexample adenocarcinoma of the pancreas, colon, ovary, lung, breast,stomach, prostate, cervix, or esophagus), sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cellcarcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor,cervical cancer, testicular tumor, bladder carcinoma, CNS tumors (suchas a glioma, astrocytoma, medulloblastoma, craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma).

Exemplary Subjects

In some examples the disclosed methods are used to treat a subject whohas a tumor, such as a tumor described herein. In some examples, thetumor has been previously treated, such as surgically or chemicallyremoved, and the disclosed methods are used subsequently to kill anyremaining undesired tumor cells that may remain in the patient.

The disclosed methods can be used to treat any mammalian subject, suchas a human, who has a tumor, such as a cancer, or has had suchpreviously removed or treated. Subjects in need of the disclosedtherapies can include human subjects having cancer, wherein the cancercells express a tumor-specific protein on their surface that canspecifically bind to the antibody-IR700 molecule. For example, thedisclosed methods can be used as initial treatment for cancer eitheralone, or in combination with radiation or other chemotherapy. Thedisclosed methods can also be used in patients who have failed previousradiation or chemotherapy. Thus, in some examples, the subject is onewho has received other therapies, but those other therapies have notprovided a desired therapeutic response. The disclosed methods can alsobe used in patients with localized and/or metastatic cancer.

In some examples the method includes selecting a subject that willbenefit from the disclosed therapies, such as selecting a subject havinga tumor that expresses a cell surface protein (such as a tumor-specificprotein) that can specifically bind to an antibody-IR700 molecule. Forexample, if the subject is determined to have a breast cancer thatexpresses HER2, the subject can be selected to be treated with ananti-HER2-IR700 molecule, such as Tra-IR700 described in Example 1, andthe subject subsequently irradiated as described herein.

Exemplary Cell Surface Proteins

In one example, the protein on the cell surface of the target cell to bekilled is not present in significant amounts on other cells. Forexample, the cell surface protein can be a receptor that is only foundon the target cell type.

In a specific example, the cell surface protein is a tumor-specificprotein (also known in the art as a tumor-specific antigen), such asmembers of the EGF receptor family (e.g., HER1, 2, 3, and 4) andcytokine receptors (e.g., CD20, CD25, IL-13R, CD5, CD52, etc.). Tumorspecific proteins are those proteins that are unique to cancer cells orare much more abundant on them, as compared to other cells, such asnormal cells. For example HER2 is primarily found in breast cancers,while HER1 is primarily found in adenocarcinomas, which can be found inmany organs, such as the pancreas, breast, prostate and colon.

Exemplary tumor-specific proteins that can be found on a target cell(and to which an antibody specific for that protein can be used toformulate an antibody-IR700 molecule), include but are not limited to:any of the various MAGEs (Melanoma-Associated Antigen E), including MAGE1 (e.g., GenBank Accession Nos. M77481 and AAA03229), MAGE 2 (e.g.,GenBank Accession Nos. L18920 and AAA17729), MAGE 3 (e.g., GenBankAccession Nos. U03735 and AAA17446), MAGE 4 (e.g., GenBank AccessionNos. D32075 and A06841.1), etc.; any of the various tyrosinases (e.g.,GenBank Accession Nos. U01873 and AAB60319); mutant ras; mutant p53(e.g., GenBank Accession Nos. X54156, CAA38095 and AA494311); p97melanoma antigen (e.g., GenBank Accession Nos. M12154 and AAA59992);human milk fat globule (HMFG) associated with breast tumors (e.g.,GenBank Accession Nos. S56151 and AAB19771); any of the various BAGEs(Human B melanoma-Associated Antigen E), including BAGE1 (e.g., GenBankAccession No. Q13072) and BAGE2 (e.g., GenBank Accession Nos. NM_182482and NP_872288), any of the various GAGEs (G antigen), including GAGE1(e.g., GenBank Accession No. Q13065) or any of GAGE2-6; variousgangliosides, CD25 (e.g., GenBank Accession Nos. NP_000408.1 andNM_000417.2).

Other tumor-specific antigens include the HPV 16/18 and E6/E7 antigensassociated with cervical cancers (e.g., GenBank Accession Nos.NC_001526, FJ952142.1, ADB94605, ADB94606, and U89349), mucin (MUC1)-KLH antigen associated with breast carcinoma (e.g., GenBank AccessionNos. J03651 and AAA35756), CEA (carcinoembryonic antigen) associatedwith colorectal cancer (e.g., GenBank Accession Nos. X98311 andCAA66955), gp100 (e.g., GenBank Accession Nos. S73003 and AAC60634)associated with for example melanoma, MARTI antigens associated withmelanoma (e.g., GenBank Accession No. NP_005502), cancer antigen 125(CA125, also known as mucin 16 or MUC16) associated with ovarian andother cancers (e.g., GenBank Accession Nos. NM_024690 and NP_078966);alpha-fetoprotein (AFP) associated with liver cancer (e.g., GenBankAccession Nos. NM_001134 and NP_001125); Lewis Y antigen associated withcolorectal, biliary, breast, small-cell lung, and other cancers;tumor-associated glycoprotein 72 (TAG72) associated withadenocarcinomas; and the PSA antigen associated with prostate cancer(e.g., GenBank Accession Nos. X14810 and CAA32915).

Other exemplary tumor-specific proteins further include, but are notlimited to, PMSA (prostate membrane specific antigen; e.g., GenBankAccession Nos. AAA60209 and AAB81971.1) associated with solid tumorneovasculature, as well prostate cancer, HER-2 (human epidermal growthfactor receptor 2, e.g., GenBank Accession Nos. M16789.1, M16790.1,M16791.1, M16792.1 and AAA58637) associated with breast cancer, ovariancancer, stomach cancer and uterine cancer, HER-1 (e.g., GenBankAccession Nos. NM_005228 and NP_005219) associated with lung cancer,anal cancer, and glioblastoma as well as adenocarcinomas; NY-ESO-1 (e.g.GenBank Accession Nos. U87459 and AAB49693) associated with melanoma,sarcomas, testicular carcinomas, and other cancers, hTERT (akatelomerase) (e.g., GenBank Accession. Nos. NM_198253 and NP_937983(variant 1), NM_198255 and NP_937986 (variant 2)); proteinase 3 (e.g.,GenBank Accession Nos. M29142, M75154, M96839, X55668, NM 00277, M96628,X56606, CAA39943 and AAA36342), and Wilms tumor 1 (WT-1, e.g. GenBankAccession Nos. NM_000378 and NP_000369 (variant A), NM_024424 andNP_077742 (variant B), NM_024425 and NP_077743 (variant C), andNM_024426 and NP_077744 (variant D)).

In one example the tumor-specific protein is CD52 (e.g., GenBankAccession. Nos. AAH27495.1 and CAI15846.1) associated with chroniclymphocytic leukemia; CD33 (e.g., GenBank Accession. Nos. NM_023068 andCAD36509.1) associated with acute myelogenous leukemia; and CD20 (e.g.,GenBank Accession. Nos. NP_068769 NP_031667) associated with Non-Hodgkinlymphoma.

Thus, the disclosed methods can be used to treat any cancer thatexpresses a tumor-specific protein.

Exemplary Antibody-IR700 Molecules

One skilled in the art will recognize that because cell surface proteinsequences are publically available (as for example shown above), thatmaking or purchasing antibodies (or other small molecules that can beconjugated to IR700) specific for such proteins is routine. For example,if the tumor-specific protein HER2 is selected as a target, antibodiesspecific for HER2 (such as Trastuzumab) can be purchased or generatedand attached to the IR700 dye. In one example, a patient is treated withat least two different antibody-IR700 molecules. In one example, the twodifferent antibody-IR700 molecules are specific for the same protein(such as HER-2), but are specific for different epitopes of the protein(such as epitope 1 and epitope 2 of HER-2). In another example, the twodifferent antibody-IR700 molecules are specific for two differentproteins or antigens, such as one antibody specific for CD4, and anotherantibody specific for CD25, which could be used for example to treat a Tcell leukemia. For example, antiHER1-IR700 and antiHER2-IR700 could beinjected together as a cocktail to facilitate killing of cells bearingeither HER1 or HER2. Other specific examples are provided in the tablebelow. In one example, the antibody is a humanized monoclonal antibody.Antibody-IR700 molecules can be generated using routine methods, such asthose described in Example 1 below. Thus, the disclosure also providesantibody-IR700 molecules, compositions that include such molecules, andkits that include such molecules (for example a kit that includes one ormore antibody-IR700 molecules and a chemotherapeutic agent, or amolecular targeting agent, or combinations thereof).

Tumor-Specific Exemplary Antibody/Small Antigen Exemplary TumorsMolecules HER1 Adenocarcinoma (e.g., Cetuximab, panitumamab, colorectalcancer, head zalutumumab, nimotuzumab, and neck cancer) matuzumab. Smallmolecule inhibitors gefitinib, erlotinib, and lapatinib can also beused. HER2 breast cancer, ovarian Trastuzumab (Herceptin ®), cancer,stomach pertuzumab cancer, uterine cancer CD20 Non-Hodgkin Tositumomab(Bexxar ®); lymphoma Rituximab (Rituxan, Mabthera); or Ibritumomabtiuxetan (Zevalin, for example in combination with yttrium-90 orindium-111 therapy) CD25 T-cell lymphoma Daclizumab (Zenapax) CD33 Acutemyelogenous Gemtuzumab (Mylotarg, for leukemia example in combinationwith calicheamicin therapy) CD52 chronic lymphocytic Alemtuzumab(Campath) leukemia CEA colorectal cancer, CEA-scan (Fab fragment, somegastric cancers, approved by FDA), colo101 biliary cancer Cancer antigenovarian cancer, OC125 monoclonal antibody 125 (CA125) mesothelioma,breast cancer Alpha- hepatocellular ab75705 (available from fetoproteincarcinoma Abcam) and other commercially (AFP) available AFP antibodiesLewis Y colorectal cancer, B3 (Humanized) biliary cancer TAG72adenocarcinomas B72.3 (FDA-approved including colorectal, monoclonalantibody) pancreatic, gastric, ovarian, endometrial, mammary, and non-small cell lung cancer Vascular Colorectal cancer Bevacizumab(Avastin ®) endothelial growth factor

Antibody-IR700 molecules for treating transplant rejection can also begenerated using Basiliximab or Daclizumab which target IL-2Rα receptor(CD25)

Administration of Antibody-IR700 Molecules and Additional TherapeuticAgents

Antibody-IR700 molecules and additional therapeutic agents (such asanti-neoplastic agents) can be contacted with a cell in vitro, forexample by adding the antibody-IR700 molecules and additionaltherapeutic to growth media in which the cells or growing, or can becontacted with a cell in vivo, for example by administering theantibody-IR700 molecules and additional therapeutic agents to thesubject to be treated.

The antibody-IR700 molecules and additional therapeutic agents can beadministered locally or systemically using any method known in the art,for example to subjects having a tumor, such as a cancer, or who has hada tumor previously removed (for example via surgery). Although specificexamples are provided, one skilled in the art will appreciate thatalternative methods of administration of the disclosed antibody-IR700molecules and additional therapeutic agents can be used. Such methodsmay include for example, the use of catheters or implantable pumps toprovide continuous infusion over a period of several hours to severaldays into the subject in need of treatment.

In one example, the antibody-IR700 molecules and additional therapeuticagents are administered by parenteral means, including direct injectionor infusion into a tumor (intratumorally). In some examples, theantibody-IR700 molecules and additional therapeutic agents areadministered to the tumor by applying the antibody-IR700 molecules andadditional therapeutic agents to the tumor, for example by bathing thetumor in a solution containing the antibody-IR700 molecules andadditional therapeutic agents or by pouring the antibody-IR700 moleculesand additional therapeutic agents onto the tumor.

In addition, or alternatively, the disclosed compositions can beadministered systemically, for example intravenously, intramuscularly,subcutaneously, intradermally, intraperitoneally, subcutaneously, ororally, to a subject having a tumor (such as cancer).

The dosages of the antibody-IR700 molecules (and additional therapeuticagents) to be administered to a subject are not subject to absolutelimits, but will depend on the nature of the composition and its activeingredients and its unwanted side effects (e.g., immune response againstthe antibody), the subject being treated and the type of condition beingtreated and the manner of administration. Generally the dose will be atherapeutically effective amount, such as an amount sufficient toachieve a desired biological effect, for example an amount that iseffective to decrease the size (e.g., volume and/or weight) of thetumor, or attenuate further growth of the tumor, or decrease undesiredsymptoms of the tumor. Dosages of additional therapeutic agents areknown in the art.

For intravenous administration of the antibody-IR700 molecules,exemplary dosages for administration to a subject for a single treatmentcan range from 0.5 to 100 mg/60 kg of body weight, 1 to 100 mg/60 kg ofbody weight, 1 to 50 mg/60 kg of body weight, 1 to 20 mg/60 kg of bodyweight, for example about 1 or 2 mg/60 kg of body weight. In yet anotherexample, a therapeutically effective amount of ip or intratumoraladministered antibody-IR700 molecules can vary from 10 μg to 5000 μg ofantibody-IR700 molecule to 1 kg of body weight, such as 10 μg/kg to 1000μg/kg, 10 μg/kg to 500 μg/kg, or 100 μg/kg to 1000 μg/kg.

In one example, the dose of antibody-IR700 molecule administered to ahuman patient is at least 50 mg, such as at least 100 mg, at least 300mg, at least 500 mg, at least 750 mg, or even 1 g.

Treatments with disclosed antibody-IR700 molecules (and additionaltherapeutic agents) can be completed in a single day, or may be donerepeatedly on multiple days with the same or a different dosage.Repeated treatments may be done on the same day, on successive days, orevery 1-3 days, every 3-7 days, every 1-2 weeks, every 2-4 weeks, every1-2 months, or at even longer intervals.

Irradiation of Cells

After the cells are contacted with one or more antibody-IR700 molecules,they are irradiated. Methods of irradiation are well known in the art.As only cells expressing the cell surface protein will be recognized bythe antibody, only those cells will have sufficient amounts of theantibody-IR700 molecules bound to it. This decreases the likelihood ofundesired side effects, such as killing of normal cells, as theirradiation will only kill the cells to which the antibody-IR700molecules are bound, not the other cells.

In some examples, cells are irradiated in vitro, such as in a tissueculture dish. In other examples, a cell is irradiated in vivo, forexample irradiating a subject who has previously been administeredantibody-IR700 molecules. In some examples, the subject is irradiated,for example a tumor in the subject can be irradiated.

The cells are irradiated with a therapeutic dose of radiation at awavelength of 660-710 nm, such as 660-700 nm, 680-7000 nm, 670-690 nm,for example, 680 nm. In particular examples, the cells are irradiated ata dose of at least 1 J cm⁻², such as at least 10 J cm⁻², at least 30 Jcm⁻², at least 50 J cm⁻², at least 100 J cm⁻², or at least 500 J cm⁻²,for example, 1-1000 J cm⁻², 1-500 J cm⁻², 30 to 50 J cm⁻², 10-100 Jcm⁻², or 10-50 J cm⁻².

Cells (or patients) can be irradiated one or more times. Thus,irradiation can be completed in a single day, or may be done repeatedlyon multiple days with the same or a different dosage (such asirradiation at least 2 different times, 3 different times, 4 differenttimes 5 different times or 10 different times). Repeated irradiationsmay be done on the same day, on successive days, or every 1-3 days,every 3-7 days, every 1-2 weeks, every 2-4 weeks, every 1-2 months, orat even longer intervals.

Exemplary Devices Containing NIR LEDs

Any type of item that can be worn or placed on the body, and is amenableto the incorporation of NIR LEDs, can be used. In one example, thedevice is a chamber into which the patient is inserted. Such devices canbe used in the treatment of tumor cells circulating in the blood orlymph, such as leukemias, lymphomas, as well as metastatic cells presentin the blood or lymph. In some examples, such devices can be used in thetreatment of tumor cells present on the skin, such as a melanoma.

To kill all the cells circulating in the body it may be necessary towear the devices for an extended period of time, such as several weeksor months. Thus, these devices can be incorporated into every dayclothing, jewelry and nightwear such as blankets. These devices make itpossible to expose the patient to NIR light using portable everydayarticles of clothing and jewelry so that treatment remains private anddoes not interfere with everyday activities. For instance, a necklaceincorporating NIR LEDs can be customizable to the patient's tastes, andworn discreetly during the day for PIT therapy (for example by killingtumor cells that pass through the carotid artery and other vasculaturein the neck). Multiple devices of a similar “everyday” nature (blankets,bracelets, necklaces, underwear, socks, shoe inserts and the like) couldbe worn by the same patient during the treatment period. For examplewhile sleeping, a patient could use the NIR blanket. The devices canalso include a power supply, such as a battery, and a cooling element toprevent overheating for such devices as blankets.

In one example, the device is jewelry, such as a ring, watch, bracelet,or necklace. In another example, the item is an article of clothing oraccessory, such as a shirt, belt, pants, underwear, socks, coat, shoeinsert, scarf, hat, wrist guard, gloves, and the like. In anotherexample, the device is an article that can cover the body, such as ablanket or towel. In another example, the device is a whole body lightchamber that exposes the skin directly (such a device could also includea power supply and/or cooling supply).

By wearing the device that incorporates one or more NIR LEDs (such as atleast 2, at least 3, at least 4, at least 5, at least 10, at least 20,or at least 50 NIR LEDs), tumor cells or other cells to be killed thatare present in the blood or lymph become exposed to the light generatedby the NIR LEDs (such as an NIR LED that emits at 660 to 740 nm, such as670 to 700 nm or 680 to 720 nm). The light emitted from the NIR LED canpenetrate the skin and blood vessels (such as the carotid artery ormicrovasculature in the skin), thus allowing the light to activate theantibody-IR700 molecule bound to the target cells, thus killing thecells to which the antibody-IR700 molecule is bound. The NIR LEDs can bearranged in the device to ensure that the skin or the blood vessels orlymphatic system are targeted.

NIR LED devices that can be used in the methods provided herein arecommercially available. The applicable products from one manufacturer,Marubeni America, are listed below. The first product, a molded LED, haslow power but it could be used over a longer exposure time. The otheroptions have higher power and thus may benefit from provisions foradditional cooling. Except for the last one, which is packaged in a 25mm×18 mm metal case, the others are applicable to wearable devices suchas bracelets, necklace, underwear, socks, gloves, hats and otherwearable items. All are usable in blankets, handheld devices orchambers.

For example, Marubeni America Corporation (tech-led.com/index.shtml)provides the following NIR LEDs with lens options to set the irradiationpattern: Molded LED (www.tech-led.com/data/L680-AU.pdf) which is 5 mm indiameter, has a total radiated power of 4 mW, calculated power densityof 5 mW cm⁻² and a power requirement of 1.8V 20 mA; Surface Mount LEDwhich is 3.5 mm×2.7 mm, has a total radiated power of 3 mW, calculatedpower density of 32 mW cm⁻², and a power requirement of 1.9V 50 mA;Super Beam (tech-led.com/Superbeam_LEDs.shtml) which is 7.6 mm×7.6 mm,has a total radiated power of 20-52 mW, calculated power density of34-90 mW cm⁻², and a power requirement of 1.65V 100 mA; High PowerSurface Mount (tech-led.com/SMB_BL_LEDs.shtml) which is 5 mm×5 mm or 7mm diameter, has a total radiated power of 90 mW, calculated powerdensity of 360 mW cm⁻² and a power requirement of 2.4V 500 mA; and HighPower Illuminators (tech-led.com/High_Power_llUuminators.shtml) which is25 mm×18 mm, has a total radiated power of 150 mW, a calculated powerdensity of 33 mW cm⁻² and a power requirement of 10V 120 mA.Alternatively, such devices can be made that emit light at 690 nm with asimilar power with short strong intermittent pulse.

During in vitro experimentation, NIR light with a power density of 2.2mW cm⁻² (or 2.2 mJ s⁻¹ cm⁻²) induced cell death. Assuming an attenuationcoefficient for tissue of 4 cm⁻¹, the intensity of the light would bedown to 10% at 5.8 mm and 1% at 12 mm. This indicates that for in vivoapplications, the power density required needs to be 10-100 timeslarger. That is, the dose of light emitted by the NIR LED device in someexamples is at least 20 mW cm⁻², such as at least 50 mW cm⁻², at least100 mW cm⁻², at least 150 mW cm⁻², at least 200 mW cm⁻² or, at least 300mW cm⁻². Multiple NIR LEDs can be arranged in a two-dimensional array tocover larger areas. In one example, a laser is used as the NIR lightsource as an alternative to an LED.

The NIR LEDs can be powered by using a power supply (which may bedirectly or indirectly part of the device). The power supply requirementwould depend on the number of LEDs in the device. For example, one ormore batteries can be used to power the NIR LED. For some LEDs, 4 AAbatteries can power 3 LEDs in series. An alkaline AA battery is rated ata maximum of 3000 mAh so this configuration provide powers for up to150, 60, and 30 hr at 20, 50 and 100 mA.

In some examples, the device further includes a cooling device (whichmay be directly or indirectly part of the device). For example, heatsinks can be used for passive or active cooling. Another alternative isa thermoelectric effect (Peltier). This would draw additional power butit can be used in applications where the power requirements would need aplug-in AC adapter.

Another type of device that can be used with the disclosed methods is aflashlight-like device with NIR LEDs. Such a device can be used forfocal therapy of lesions during surgery, or incorporated into endoscopesto apply NIR light to body surfaces after the administration of PITagent. Such devices can be used by physicians or qualified healthpersonnel to direct treatment to particular targets on the body.

Treatment Using Wearable NIR LEDs

As described herein, the disclosed methods are highly specific forcancer cells. However, in order to kill the cells circulating in thebody or present on the skin, the patient can wear a device thatincorporates an NIR LED. In some example the patient uses at least twodevices, for example an article of clothing or jewelry during the day,and a blanket at night. In some example the patient uses at least twodevices at the same time, for example two articles of clothing. Thesedevices make it possible to expose the patient to NIR light usingportable everyday articles of clothing and jewelry so that treatmentremains private and does not interfere with everyday activities. In someexamples, the device can be worn discreetly during the day for PITtherapy.

In one example, the patient is administered one or more antibody-IR700molecules, using the methods described herein. The patient then wears adevice that incorporates an NIR LED, permitting long-term therapy andtreatment of tumor cells that are present in the blood or lymph or onthe skin. In some examples, the dose is at least at least 1 J cm⁻², atleast 10 J cm⁻², at least 20 J cm⁻², or at least 30 J cm⁻², such as 20 Jcm⁻² or 30 J/cm². In some examples, administration of the antibody-IR700molecule is repeated over a period of time (such as bi-weekly ormonthly, to ensure therapeutic levels are present in the body.

In some examples, the patient wears or uses the device, or combinationof devices, for at least 1 week, such as at least 2 weeks, at least 4weeks, at least 8 weeks, at least 12 weeks, at least 4 months, at least6 months, or even at least 1 year. In some examples, the patient wearsor uses the device, or combination of devices, for at least 4 hours aday, such as at least 12 hours a day, at least 16 hours a day, at least18 hours a day, or 24 hours a day. It is quite possible that multipledevices of a similar “everyday” nature (blankets, bracelets, necklaces,underwear, socks, shoe inserts) could be worn by the same patient duringthe treatment period. At night the patient can use the NIR LED blanketor other covering.

Additional Treatments

As discussed above, prior to, during, or following administration of oneor more antibody-IR700 molecules, the subject can receive one or moreother therapies. In one example, the subject receives one or moretreatments to remove or reduce the tumor prior to administration of theantibody-IR700 molecules.

Examples of such therapies that can be used in combination with thedisclosed PIT methods, which enhance accessibility of the tumor toadditional therapeutic agents for about 8 hours after the PIT, but arenot limited to, surgical treatment for removal or reduction of the tumor(such as surgical resection, cryotherapy, or chemoembolization), as wellas anti-tumor pharmaceutical treatments which can includeradiotherapeutic agents, anti-neoplastic chemotherapeutic agents,antibiotics, alkylating agents and antioxidants, kinase inhibitors, andother agents. In some examples, the additional therapeutic agent isconjugated to a nanoparticle. Particular examples of additionaltherapeutic agents that can be used include microtubule binding agents,DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and/orRNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors,and gene regulators. These agents (which are administered at atherapeutically effective amount) and treatments can be used alone or incombination. Methods and therapeutic dosages of such agents are known tothose skilled in the art, and can be determined by a skilled clinician.

“Microtubule binding agent” refers to an agent that interacts withtubulin to stabilize or destabilize microtubule formation therebyinhibiting cell division. Examples of microtubule binding agents thatcan be used in conjunction with the disclosed antibody-IR700 moleculetherapies include, without limitation, paclitaxel, docetaxel,vinblastine, vindesine, vinorelbine (navelbine), the epothilones,colchicine, dolastatin 15, nocodazole, podophyllotoxin and rhizoxin.Analogs and derivatives of such compounds also can be used and are knownto those of ordinary skill in the art. For example, suitable epothilonesand epothilone analogs are described in International Publication No. WO2004/018478. Taxoids, such as paclitaxel and docetaxel, as well as theanalogs of paclitaxel taught by U.S. Pat. Nos. 6,610,860; 5,530,020; and5,912,264 can be used.

The following classes of compounds can be used with the PIT methodsdisclosed herein: suitable DNA and/or RNA transcription regulators,including, without limitation, actinomycin D, daunorubicin, doxorubicinand derivatives and analogs thereof also are suitable for use incombination with the disclosed therapies. DNA intercalators andcross-linking agents that can be administered to a subject include,without limitation, cisplatin, carboplatin, oxaliplatin, mitomycins,such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide andderivatives and analogs thereof. DNA synthesis inhibitors suitable foruse as therapeutic agents include, without limitation, methotrexate,5-fluoro-5′-deoxyuridine, 5-fluorouracil and analogs thereof. Examplesof suitable enzyme inhibitors include, without limitation, camptothecin,etoposide, formestane, trichostatin and derivatives and analogs thereof.Suitable compounds that affect gene regulation include agents thatresult in increased or decreased expression of one or more genes, suchas raloxifene, 5-azacytidine, 5-aza-2′-deoxycytidine, tamoxifen,4-hydroxytamoxifen, mifepristone and derivatives and analogs thereof.Kinase inhibitors include Gleevac, Iressa, and Tarceva that preventphosphorylation and activation of growth factors.

Other therapeutic agents, for example anti-tumor agents, that may or maynot fall under one or more of the classifications above, also aresuitable for administration in combination with the disclosed PITtherapies. By way of example, such agents include adriamycin, apigenin,rapamycin, zebularine, cimetidine, and derivatives and analogs thereof.

In some examples, the subject receiving the therapeutic antibody-IR700molecule composition is also administered interleukin-2 (IL-2), forexample via intravenous administration. In particular examples, IL-2(Chiron Corp., Emeryville, Calif.) is administered at a dose of at least500,000 IU/kg as an intravenous bolus over a 15 minute period everyeight hours beginning on the day after administration of the peptidesand continuing for up to 5 days. Doses can be skipped depending onsubject tolerance.

In some examples, the disclosed antibody-IR700 molecules can beco-administered (or administered shortly before or after theirradiation) with a fully human antibody to cytotoxic T-lymphocyteantigen-4 (anti-CTLA-4). In some example subjects receive at least 1mg/kg anti-CTLA-4 (such as 3 mg/kg every 3 weeks or 3 mg/kg as theinitial dose with subsequent doses reduced to 1 mg/kg every 3 weeks).

In one example, at least a portion of the tumor (such as a metastatictumor) is surgically removed (for example via cryotherapy), irradiated,chemically treated (for example via chemoembolization) or combinationsthereof, prior to administration of the disclosed therapies (such asadministration of antibody-IR700 molecules). For example, a subjecthaving a metastatic tumor can have all or part of the tumor surgicallyexcised prior to administration of the disclosed therapies. In anexample, one or more chemotherapeutic agents are administered followingtreatment with antibody-IR700 molecules and irradiation. In anotherparticular example, the subject has a metastatic tumor and isadministered radiation therapy, chemoembolization therapy, or bothconcurrently with the administration of the disclosed therapies.

Example 1 Synthesis of IRDye 700-Conjugated Trastuzumab (Anti-Her2)

This example describes methods used to conjugate the monoclonal antibodyTrastuzumab to the IRDye 700DX NHS Ester. On skilled in the art willrecognize that any antibody, such as any monoclonal antibody specificfor a target cell surface protein, can be conjugated to IRDye 700DX NHSEster using similar methods.

Humanized anti-HER2 antibody, Trastuzumab (Tra; Genentech, SanFrancisco, Calif.) (1 mg, 6.8 nmol) was incubated with IRDye 700DX NHSEster (IR700; LI-COR Bioscience, Lincoln, Nebr.) (66.8 μg, 34.2 nmol, 5mmol/L in DMSO) in 0.1 mol/L Na₂HPO₄ (pH 8.5) at room temperature for 30to 120 min. Trastuzumab is a recombinant humanized monoclonal antibody(mAb) directed against the extracellular domain of the human epidermalgrowth factor receptor (EGFR) 2 (HER2) tyrosine kinase receptor. Themixture was purified with a Sephadex G50 column (PD-10; GE Healthcare,Piscataway, N.J.). The protein concentration was determined withCoomassie Plus protein assay kit (Pierce Biotechnology, Rockford, Ill.)by measuring the absorption at 595 nm with a UV-Vis system (8453 ValueSystem; Agilent Technologies, Palo Alto, Calif.). The concentration ofIR700 was measured by absorption with the UV-Vis system to confirm thenumber of fluorophore molecules conjugated to each Trastuzumab molecule.The number of IR700 per Trastuzumab was about 3.

The purity of the Tra-IR700 conjugate was confirmed by analyticalsize-exclusion HPLC (SE-HPLC) and sodium dodecyl sulfatepolyacrylamidegel elctrophoresis (SDS-PAGE). SE-HPLC was performed usinga Beckman System Gold (Fullerton, Calif.) equipped with model 126solvent delivery module, a model 168 UV detector, and a JASCOfluorescence detector (excitation 689 nm and emission at 700 nm)controlled by 32 Karat software. SE chromatography was performed on aTSKgel G2000SWx1 (Tosoh Bioscience LLC, Montgomeryville, Pa.) eluted for45 minutes using phosphate buffered saline (PBS) at 0.5 mL/min. SDS-PAGEwas performed with a 4% to 20% gradient polyacrylamide gel (Invitrogen,Carlsbad, Calif.). Just after separating the proteins, fluorescenceintensity was analyzed with a Fujifilm FLA-5100 fluorescence scanner(Valhalla, N.Y.) with an internal laser of 670 nm for excitation and 705nm long pass filter for emission. The fluorescence intensity of eachband was analyzed with Multigage software (Fujifilm). The gels were thenstained with Colloidal Blue Staining Kit (Invitrogen), and digitallyscanned. The protein concentration in each band was analyzed with ImageJsoftware. The trastuzumab-1R700 (Tra-1R700) and panitumumab-1R700(Pan-1R700; see Example 8) preparations demonstrated strong associationand contained no detectable MAb aggregates as determined by highperformance liquid chromatography (HPLC) and sodium dodecyl sulfatepolyacrylamidegel electrophoresis SDS-PAGE.

To determine the in vitro binding characteristics of IR700 conjugates¹²⁵I labeling of the conjugates using the Indo-Gen procedure wasperformed. The specific activities of the radiolabeled antibodies were8.52 mCi/mg for Trastuzumab and 7.84 mCi/mg for panitumumab (see Example8 below). It was observed that 73.38±0.39% (¹²⁵I-Tra-IR700) and78.61±0.89% (¹²⁵I-Pan-IR700) of binding was achieved with each MAbconjugate respectively and the specificity of binding was confirmed byblocking with excess native unconjugated MAb (less than 1.4%). Sinceimmunoreactivity of ¹²⁵I-Tra and ¹²⁵I-Pan measured with the same methodwere 78±2%, and 82±3%, respectively, minimal loss of MAbs with IR700conjugation was confirmed. Immunoreactivity assay was performed asdescribed previously. Briefly, after trypsinization, 2×10⁶ of 3T3/HER2or A431 cells were resuspended in PBS containing 1% bovine serum albumin(BSA). ¹²⁵I-Tra-IR700 or ¹²⁵I-Pan-IR700 (1 mCi, 0.2 μg) was added andincubated for 1 h on ice. Cells were washed, pelleted, the supernatantdecanted, and counted in a 2470 Wizard² γ-counter (Perkin Elmer,Shelton, Conn.). Nonspecific binding to the cells was examined underconditions of antibody excess (200 μg of nonlabeled trastuzumab orpanitumumab).

Example 2 Selective Killing of HER2+ Cells

This example describes methods used to show that the Trastuzumab-IR700compound described in Example 1 (referred to herein as Tra-IR700) can beused to selectively kill cells that express HER2 (HER2+), but hasminimal negative effects on HER2 negative (HER2−) cells.

HER2 gene-transfected NIH3T3 (3T3/HER2+) cells were used for targetphotodynamic therapy (PDT). As a control, Balb/3T3 cells which expressDsRed fluorescent protein but not HER2 (Balb/3T3/DsRed) were employed.Cells were grown in RPMI 1640 supplemented with 10% fetal bovine serumand 1% penicillin/streptomycin in tissue culture flasks in a humidifiedincubator at 37° C. in an atmosphere of 95% air and 5% carbon dioxide.

Fluorescence microscopy was performed with a BX51 or IX81 microscope(Olympus America, Melville, N.Y.). The filter was set to detect IR700and consisted of a 590-650 nm excitation filter, and a 665-740 nm bandpass emission filter. To detect DsRed protein, a filter set consistingof a 480-550 nm excitation filter, and a 590 nm long pass emissionfilter was employed.

Fluorescence microscopy was performed to test the subcellularlocalization of IR700 in 3T3/HER2+ cells. Cells were seeded on coverglass-bottomed dishes and incubated for 24 hours. Tra-IR700 was added tothe culture medium at 10 μg/mL. As shown in FIG. 1A, Tra-IR700 wasdetected on the cell surface after 1 hour incubation on ice, and wasmainly localized to the lysosome 6 hours after incubation at 37° C.,indicating gradual internalization. Co-staining with LysoTracker Green(Invitrogen, Carlsbad, Calif.), which was detected by a filter setconsisting of a 420-480 nm excitation filter, and a 520 nm long passemission filter, revealed co-localization of IR700 with theendolysosomal compartment (FIG. 1B). After 1 h and 6 h of incubationwith Tra-1R700, excitation light (fluorescence microscope; power densityof 2.2 mW cm-2) induced fluorescence as well as cellular swelling, blebformation, and rupture of vesicles representing necrotic cell death(FIG. 1C).

For photoimmunotherapy (PIT), cells were seeded on 35 mm coverglass-bottomed dishes and incubated 24 hours. Medium was replaced withfresh culture medium containing Tra-IR700 at 10 μg/mL and incubated for6 hours at 37° C. Culture medium was replaced with phenol red-freemedium after washing with phosphate buffered saline (PBS). Cells wereirradiated with light at 670 nm to 690 nm using a red light emittingdiode (LED; FluorVivo; INDEC Systems Inc., Capitola, Calif.) with apower density of 2.6 mW cm⁻² as measured with optical power meter (PM100, Thorlabs, Newton, N.J.). Cell viability was assessed 1 hour aftertreatment with LIVE/DEAD@ Fixable Green Dead Cell Stain Kit(Invitrogen). After the treatment, cells were trypsinized and washedwith PBS. Green fluorescent reactive dye was added in the cellsuspension and incubated at room temperature for 30 minutes. Cells werethen analyzed on flow cytometer (FACS Calibur, BD BioSciences, San Jose,Calif.).

As shown in FIG. 1C, irradiation at 1.0 J cm-2 for 3T3/HER2+ cellsresulted in rapid necrotic cell death, representing budding and swellingof the cell membrane.

Example 3 Identification of Irradiation Dose

To determine the phototoxicity in response to different doses of lightirradiation, PDT-treated 3T3/HER2+ cells were assayed by flow cytometryusing the LIVE/DEAD® Fixable Green Dead Cell Stain Kit. The LIVE/DEADassay, which can detect the cells with damaged membranes, was performedwithin 1 h after the treatment. As shown in FIG. 1D, cell death inresponse to Tra-IR700-mediated PDT was light dose dependent 1 hour afterPIT. Cells without PIT or Tra-IR700 did not show significantphototoxicity.

Example 4 Measurement of Cell Viability Over Time

To monitor cell viability over time, cells were labeled and irradiatedas described in Example 2, then were monitored subsequently over time (5days) using microscopy as described in Example 2.

As shown in FIG. 1F, phototoxic cell death was observed only in thetreated 3T3/HER2+ cells with Tra-IR700, but not in the un-irradiatedgroup (no PIT) or the group irradiated but did not receive Tra-IR700 (NoTra-IR700).

Example 5 Target Specific Phototoxicity of Tra-IR700

PIT was performed as described in Example 2. As shown in FIG. 1G, therewas no significant difference in phototoxicity between 1 h and 6 hincubation with Tra-1R700, indicating that membrane binding of Tra-1R700was sufficient to induce cell death. When Tra-1R700 was localized to theendolysosomal compartment (FIG. 1B), it also induced rupture of thevesicle with cellular swelling and bleb formation after irradiation.However, this did not appear to be a major cause of cell death, sincecell death was observed without endolysosomal localization of Tra-IR700within 1 h of incubation at 4° C. Failure to wash the cells prior toirradiation did not influence the phototoxic effect, indicating thatcellular membrane binding was important to the phototoxic effects of theconjugate, not merely the presence of the conjugate. Further, the IR700dye alone (200 nM; equivalent IR700 concentration of Tra-1R700conjugates) did not incorporate into the cells or induce phototoxcity incells (FIG. 1H and FIG. 2B). Additionally, phototoxcity wasdose-dependently blocked by the excess unconjugated trastuzumab (FIGS.2C and 2D). Furthermore, Tra-1R700 did not induce therapeutic effect toA431 cells (FIG. 1I). These results confirm that cell death is dependenton specific membrane binding of Tra-1R700.

Reactive oxygen species (ROS) have been implicated in the cell deathassociated with conventional PDT. To clarify the role of photon-inducedredox reactions (e.g. singlet oxygen (¹0₂)) in producing phototoxicitywith Tra-1R700, a redox quencher, sodium azide (NaN₃), was added to themedium when cells were irradiated. The percentage of cell death waspartially decreased in the presence of sodium azide, in a dose dependentmanner (FIG. 1J).

To confirm that the phototoxicity was target specific, 3T3/HER2+ cellsand Balb/3T3/DsRed cells (a parental HER2 negative Balb/3T3 transfectedwith DsRed fluorescent protein) were co-cultured, and irradiated at 1.0J cm⁻² after 1 or 6 hours of incubation with Tra-IR700 at 37° C.Tra-1R700 was distributed in a HER2 specific manner while DsRedexpressing Balb/3T3 cells did not show phototoxicity upon irradiation(FIG. 3A). In addition, LIVE/DEAD Green staining demonstrated HER2specific induction of cell death as determined by multi-colorfluorescence microscopy (FIG. 3B) and flow cytometry analysis (FIG. 3C).

Example 6 Tra-IR700 Reduces Proliferation of HER2+ Cells

3T3/HER2 cells were seeded into 35 mm cell culture dishes at a densityof 1×10⁴. The next day, cells were incubated with or without Tra-IR700and irradiated as described in Example 2. Cell viability was determinedby trypan blue dye exclusion assay at day 1, 3 and 5 after the cellseeding. Viable cells for treated or untreated controls were counted ona hemacytometer after trypsinization. Cell growth was also photographedunder microscope at each time point.

As shown in FIG. 1E, cells treated with Tra-IR700 and then subjected toPDT 2.0 J cm⁻² had significantly reduced viability as compared to cellsonly treated with Tra-IR700, only PDT, or no treatment.

Example 7 Tra-IR700 Selectively Kills HER2+ Cells In Vivo

This example describes methods used to show that Tra-IR700 can treatHER2+ tumors in vivo. One skilled in the art will appreciate thatsimilar methods can be used with other tumor/antibody-IR700combinations.

Six- to eight-week-old female homozygote athymic nude mice (CharlesRiver, NCI-Frederick, Frederick, Md.) were anesthetized with isoflurane.3T3/HER2+ or Balb/3T3 cells (2 million) were injected subcutaneously inthe left dorsum of the mice. Four days after cell injection, either 50or 300 μg of Tra-IR700 was administered intravenously. Her2-specificTra-IR700 accumulation in tumor xenografts was confirmed with an in vivofluorescence imaging system (Pearl Imager, LI-COR Biosciences, Lincoln,Nebr.). 3T3/HER2+ tumor-specific IR700-Tra localization was observed. Incontrast, Balb/3T3 tumor did not show Her2-specific IR700 fluorescence.

To evaluate the efficacy of targeted PIT with IR700-Tra in vivo, 1million 3T3/HER2+ or Balb/3T3 cells were injected subcutaneously intothe bilateral dorsum of female nude mice. When the volume of both tumorsreached ˜70 mm³ (about 4 days), animals were randomized into five groupsof at least 12 animals per group for the following treatments: (1) notreatment; (2) 300 μg of trastuzumab injected intravenously, no PIT; (3)300 μg of Tra-IR700 injected intravenously, no PIT; (4) PIT at 50 J/cm²for 3T3/HER2 tumor without Tra-IR700; (5) 300 μg of Tra-IR700 injectedintravenously, PDT was performed at 50 J/cm².

Twenty-four hours after administration of Tra-IR700, in mice receivingPIT a 1 cm-diameter area encompassing the right dorsum including thetumor was irradiated (dose level 50 J cm⁻²). The left dorsum of thetumor was covered with black tape to prevent its exposure to light.Effects in response to PIT were monitored daily and tumor volumes weremeasured with a caliper twice a week until it reached 500-1000 mm³, atwhich time mice were euthanized with carbon dioxide gas. In order todetermine tumor volume, the greatest longitudinal diameter (length) andthe greatest transverse diameter (width) were determined with externalcaliper.

Tumor volume based on caliper measurements were calculated by thefollowing formula; tumor volume=length×width²×0.5. Data are expressed asmeans+sem from a minimum of three experiments, unless otherwiseindicated. Statistical analyses were carried out using a statisticsprogram (GraphPad Prism; GraphPad Software, La Jolla, Calif.). Formultiple comparisons, a one-way analysis of variance (ANOVA) with posttest (Kruskal-Wallis test with post-test) was used. The cumulativeprobability of survival, determining herein as the tumor volume failedto reach 500 mm³, were estimated in each group with the use of theKaplan-Meier survival curve analysis, and the results were compared withuse of the log-rank test with Bonferroni's correction for multiplicity.P<0.05 was considered to indicate a statistically significantdifference.

As shown in FIGS. 4A and 4B, whereas 50 J cm⁻² irradiation resulted insignificant tumor growth inhibition in 3T3/HER2+ tumors at day 4, 7, 10and 14 days after treatment, untreated tumors did not exhibit anydetectable effect on tumor growth. In addition, irradiation for Balb/3T3tumors did not show significant therapeutic effect. Furthermore, nolethal side effects were found during or after the treatment.

Example 8 Synthesis of IRDye 700-Conjugated Vectibix® (Anti-HER1)

Panitumumab (Vectibix®), a fully humanized IgG2 MAb directed against thehuman EGFR was purchased from Amgen (Thousand Oaks, Calif.) andconjugated to IR700 using the methods described in Example 1. Thiscompound is referred to as Panitumumab-IR700 or Pan-IR700. The number ofIR700 per Panitumumab was about 3.

Example 9 Pan-IR700 Selectively Kills HER1+ Cells

This example describes methods used to show that the Pan-IR700 compounddescribed in Example 8 can be used to selectively kill cells thatexpress HER1 (HER1+), but has minimal negative effects on HER1 negative(HER1−) cells.

EGFR-expressing A431 cells were used as the target HER1+ cells. As acontrol, Balb/3T3 cells which express DsRed fluorescent protein but donot express HER1/EGFR (Balb/3T3/DsRed) were used. Cells were grown inRPMI 1640 supplemented with 10% fetal bovine serum and 1%penicillin/streptomycin in tissue culture flasks in a humidifiedincubator at 37° C. in an atmosphere of 95% air and 5% carbon dioxide.A431 or Balb/3T3/DsRed cells were seeded on cover glass-bottomed dishesand incubated for 24 hours. Pan-IR700 was added to the culture medium at10 μg/mL and incubated either for 1 hour on ice or 6 hours at 37° C.,then cells were washed with PBS. Culture medium was replaced with phenolred-free medium after washing with phosphate buffered saline (PBS).

Fluorescence microscopy was performed as described in Example 2 todetect the antigen-specific localization of IR700. Pan-IR700 wasdetected on the cell surface of A431 cells after 1 hour incubation onice, and was mainly localized to the lysosome 6 hours after incubationat 37° C. No significant IR700 signal was observed with Balb/3T3/DsRedcells.

PIT was performed as described in Examples 2 and 3. As shown in FIG. 5A,irradiation of A431 cells at 0.5 to 2 J cm⁻² resulted in rapid celldeath in a dose-dependent manner, representing budding and swelling ofthe cell membrane. As shown in FIG. 5B, the percentage of cell death intarget cells versus untreated control cells was significantly influencedby excitation light dose. In addition, there was no significantcytotoxicity associated with exposure to Pan-1R700 without excitationlight or with light exposure without Tra-1R700. However, panitumumabitself had a noticeable treatment effect against A431 cells due to downregulation and signal inhibition of HER1 (Yang et al., Cancer Res59:1236-43, 1999).

Target-specific phototoxicity was also confirmed with Pan-1R700 mediatedPIT in A431 cells and Balb/3T3/DsRed (HER1 negative) co-cultured cells(FIG. 5C). In summary, Tra-1R700 and Pan-1R700 showed identicaltherapeutic effects to HER2 positive (3T3/HER2) and HER1 positive (A431)cells, respectively, except that unconjugated panitumumab showednoticeable growth inhibition but unconjugated trastuzumab did not reducegrowth with the dose used.

Example 10 Pan-IR700 Selectively Kills HER1+ Cells In Vivo

This example describes methods used to show that Pan-IR700 can treatHER1+ tumors in vivo. One skilled in the art will appreciate thatsimilar methods can be used with other tumor/antibody-IR700combinations.

Six- to eight-week-old female homozygote athymic nude mice (CharlesRiver, NCI-Frederick, Frederick, Md.) were anesthetized with isoflurane.One million A431 cells were injected subcutaneously in the left dorsumof the mice. Four days after cell injection, either 50 or 300 μg ofPan-IR700 was administered intravenously.

To confirm antigen specific localization of Pan-IR700, 1×10⁶ of 3T3/HER2cells (HER1 negative) were injected subcutaneously in the right dorsumat the same time of A431 cells injection. Fluorescence images wereobtained at indicated time point with Pearl Imager (LI-COR Biosciences)using 700 nm fluorescence channel. Regions of interest (ROI) for bothtumor and background were placed for equivalent sized areas containingthe same number of pixels. Tumor to background ratio (TBR) wascalculated using the following formula: TBR=((mean tumorintensity)−(mean background intensity))/((mean non−tumorintensity)−(mean background intensity)).

As shown in FIG. 6A, Pan-IR700 localized to the A431 tumor. Thefluorescence intensity of Pan-1R700 in a A431 tumor decreased graduallyover days, while tumor to background ratios (TBRs) increased (FIGS. 6Band 6C). The fluorescence intensity of the 3T3/HER2 tumor was the sameas that of background (non-tumor lesions). When 300 μg of Pan-1R700 wasadministered intravenously, fluorescence intensity of the A431 tumor wasmore than 3 times higher than 50 μg injection at 1 day after injection,however, TBR was lower because of high background signal (FIGS. 6B and6C). As less antitumor activity was found in mice receiving 50 μg (vs.300 μg) of Pan-1R700 injection following irradiation the higherinjection dose was used (FIG. 6H). Biodistribution of Tra-1R700 wasdetermined with IR700 fluorescence because tissue levels ofradioactivity and fluorescence might be different due to their differentexcretion routes and catabolism when using dual-labeledradiolabeled-Pan-1R70015. There was no other specific localization ofIR700 except for bladder accumulation on day 1 probably due to excretionof catabolized and unbound dye (FIG. 6d ).

As shown in FIG. 6D, PIT treatment following Pan-IR700 administrationbegan to shrink tumors at day 2, in contrast to non-PIT treated tumorswhich did not shrink.

To determine the effect of Pan-IR700 or carrier alone followed by PIT,the following methods were used. In order to determine tumor volume, thegreatest longitudinal diameter (length) and the greatest transversediameter (width) were determined with external caliper. Tumor volumebased on caliper measurements were calculated by the following formula;tumor volume=length×width²×0.5². Four days after A431 cell injection asdescribed above, tumor volume reaching around 40 mm³ were selected forthe study. Animals were randomized into 8 groups of at least 12 animalsper group for the following treatments: (1) no treatment; (2) 300 μg ofpanitumumab injected intravenously, no PIT; (3) 300 μg Pan-IR700injected intravenously, no PIT; (4) PIT was performed at 30 J/cm²without Pan-IR700; (5) Free IR700 dye, dose equivalent to 300 μg ofPan-IR700, was injected intravenously, and PIT was performed at 30 Jcm⁻²; (6) 50 μg of Pan-IR700 was injected intravenously, PIT wasperformed at 30 J cm⁻²; (7) 50 μg of Pan-IR700 and 250 μg of panitumumabwas injected intravenously, PIT was performed at 30 J cm⁻²; and (8) 300μg of Pan-IR700 was injected intravenously, PIT was performed at 30 Jcm⁻². After the treatment mice were monitored daily, and tumor volumewas measured twice a week until the tumor volume reached 500 mm³, atwhich time mice were euthanized with carbon dioxide gas. To test ashort-term toxicity, 300 μg of Pan-IR700 was repeatedly administratedintravenously for non-tumor-bearing mice, twice a week, for 4 weeks.

As shown in FIG. 6E, whereas treatment with Pan-IR700 and 30 J cm⁻²irradiation resulted in significant tumor growth inhibition in A431(HER1+) tumors at day 3, 7, 10, 14 and 17 days after treatment,untreated tumors did not exhibit any detectable effect on tumor growth.In addition, as shown in FIG. 6F, treatment with Pan-IR700 and 30 J cm⁻²irradiation resulted in significant increases in survival time of micewith A431 (HER1+) tumors. Furthermore, no lethal side effects were foundduring or after the treatment. FIG. 6G shows microscopic images of cellsfour days following treatment with Pan-IR700 followed by no PIT therapyor PIT therapy. Pathological analysis revealed that only scant viableA431 tumor cells were present after Pan-IR700 mediated PIT and massivegranulation with inflammatory change was observed in the tumor nodule.It was also observed that tissue edema developed superficially. Toassess the acute phase toxicity of Pan-1R700, we repeatedlyadministrated 300 μg of Pan-1R700 intravenously twice a week for 4weeks, but there were no adverse effects observed up to 8 w (n=4)compared with the control group.

Example 11 HuJ591-IR700 Selectively Kills PSMA+ Cells In Vivo

This example describes methods used to show that HuJ591-IR700 can treatprostate-specific membrane antigen (PSMA)+ tumors (such as those foundin prostate cancer) in vivo. One skilled in the art will appreciate thatsimilar methods can be used with other tumor/antibody-IR700combinations.

J591, a fully humanized IgG2 MAb directed against human PSMA wasobtained from Prof. Neil Bander, Cornell Univ and conjugated to IR700using the methods described in Example 1. This compound is referred toas J591-IR700. The number of IR700 per J591 was about 2.

Six- to eight-week-old female homozygote athymic nude mice (CharlesRiver, NCI-Frederick, Frederick, Md.) were anesthetized with isoflurane.On day 0 Two million PC3-PIP cells (PSMA+) were injected subcutaneouslyin the bottom dorsum of the mice and PC3-FLU cells (PSMA−) were injectedsubcutaneously in the top dorsum of the mice. On day 3, 100 μg ofPSMA-IR700 was administered ip.

To confirm antigen specific localization of J591-IR700, 2×10⁶ of PC3-FLUcells (PSMA−) were injected subcutaneously in a different area at thesame time of PC3-PIP cell injection. Fluorescence images were obtainedat indicated time point with Pearl Imager (LI-COR Biosciences) using 700nm fluorescence channel, as described in Examples 7 and 10.

As shown in FIG. 7, J591-IR700 localized to the PC3-PIP tumor.Nonspecific blood pool and enhanced permeability and retention effects(EPR effect) dimly show PC3-FLU (PSMA−) tumor.

To determine the effect of HuJ591-IR700 in the presence or absence ofPIT, the right side of the mouse was irradiated and the left side wasnot, as described in Examples 7 and 10. Specifically, on day 4, micereceived PIT 50 J/cm² for right tumors, on day 5 PIT 100 J/cm² for righttumors (and an image obtained), on day 11, J591-IR700 was administered(100 μg ip) and an image obtained, on day 12 PIT 50 J/cm² for righttumors, on day 13 PIT 100 J/cm² for right tumors and on day 19J591-IR700 was administered (100 μg ip). On Day 20, an image wasobtained and the tumor excised and imaged. As shown in FIG. 7, PITtreatment following J591-IR700 administration began to shrink tumors atday 5, in contrast to non-PIT treated tumors which did not shrink.

Example 12 Selective Killing In Vitro by Antibody-IR700 Molecules

This example describes additional results showing that the disclosedantibody-IR700 compounds selectively kill cells that express theappropriate protein. The photoimmunotherapy (PIT) methods are describedin Example 2.

As shown in FIG. 8, Tra-1R700 specifically killed HER2 expressing3T3/1-IER2, SHAW, SKOV3 and MDA-MB-453 cells, Pan-1R700 specificallykilled HER1 expressing A431 and MDA-MB-468 cells, and huJ591-1R700specifically killed prostate specific membrane antigen (PSMA) expressingLNCaP cells.

Example 13 Trastuzumab-IR700 Treatment of Metastases

This example describes methods used to show that Tra-IR700 can treatlung metastases.

HER2 expressing 3T3/HER2 cells (0.5 to 2 million cells) were injectedintravenously into tail vein of female nude mice. Trastuzumab-IR700 (100μg) was injected intravenously 5 days after the tumor cell injection. Asmultiple tiny lung metastases were confirmed with Tra-IR700 localizationin ex vivo imaging, the lungs were treated with 30 J/cm2 of NIR lightfrom outside the body 2 days after Trastuzumab-IR700 injection. It wasobserved that the lung metastases cleared, and there was an observedincrease in the overall survival time of the mice as compared to micethat did not receive Tra-IR700.

Example 14 Real-Time Monitoring of In Vivo Acute Necrotic Cancer CellDeath

This example describes methods used to monitor in vivo acute necroticcancer cell death induced by near infrared photoimmunotherapy inreal-time using fluorescence lifetime imaging. Although a specificexample of Pan-IR700 is described, one will appreciate that otherantibody-IR-700 molecules can be used for other tumors.

As described herein, monoclonal antibody-based, highly specificphototherapy (photoimmunotherapy; PIT) that utilizes a near infrared(NIR) phthalocyanine dye, IRDye700DX (IR700) conjugated with mAbs. NIRlight exposure leads to immediate, target-selective necrotic cell deathin vitro. Detecting immediate in vivo cell death is more difficultbecause it takes at least three days for the tumor to begin to shrink insize. In this example, fluorescence lifetime (FLT) was evaluated beforeand after PIT for monitoring the immediate cytotoxic effects of NIRmediated mAb-1R700 PIT. Anti-EGFR panitumumab-IR700 was used fortargeting EGFR-expressing A431 tumor cells. PIT with various doses ofNIR light was performed in cell pellets in vitro and in subcutaneouslyxenografted tumors in mice in vivo. FLT measurements were obtainedbefore and 0, 6, 24 and 48 h after PIT. in vitro, PIT at higher doses ofNIR light immediately led to greater FLT shortening in A431 cells. invivo, PIT induced immediate shortening of FLT in treated tumors after athreshold NIR dose of 30 J/cm² or greater. In contrast, lower levels ofNIR light (10 J/cm² or smaller) did not induce shortening of FLT. Basedon these observations, FLT imaging can be used to monitor the early andmassive cytotoxic effects of mAb-1R700-induced PIT even beforemorphological changes can be seen in the targeted tumors.

Materials and Methods Reagents.

Panitumumab, a fully humanized IgG2 monoclonal antibody (MAb) directedagainst the human EGFR, or HER1, was purchased from AMGEN Inc. A watersoluble, silicon-phthalocyanine derivative, IRDye 700DX NHS ester(IR700; C74H96N12Na4027S6Si3, molecular weight of 1954.22) was purchasedfrom LI-COR Bioscience. All other chemicals used were of reagent grade.

Synthesis of 1R700-Conjugated Panitumumab.

Panitumumab (1 mg, 6.8 nmol) was incubated with IR700 (66.8 μg, 34.2nmol, 5 mmol/L in DMSO) in 0.1 mollL Na₂HPO₄ (pH 8.6) at roomtemperature for 1 h. Then the mixture was purified with a Sephadex G50column (PD-10; GE Healthcare). The protein concentrations weredetermined with Coomassie Plus protein assay kit (Pierce Biotechnology)by measuring light absorption at 595 nm (8453 Value System; AgilentTechnologies). The concentration of IR700 was measured by absorptionwith spectroscopy to confirm the average number of fluorophore moleculesconjugated to each Panitumumab molecule. The number of IR700 perantibody was approximately 4 for the 1:4.5 reaction conditions. Theaddition of 0.4% SDS to the sample dissociated the fluorophores fromeach other, effectively causing dequenching. Quenching efficiency (QE)for a particular conjugation is defined as the fluorescence intensitywith SDS divided by fluorescence intensity without SDS.Panitumumab-1R700 conjugate (Pan-1R700) demonstrated a QE of about 4.0at pH 7.2. Pan-1R700 was kept at 4° C. in the refrigerator as a stocksolution.

Fluorescence Lifetime Measurements.

FLT experiments were performed with the eXplore OptixTm-MX2 system (ARTAdvanced Research Technologies, Inc.) (Hutchinson et al., Biophys J68:1574-82, 1995; Ma et al., Appl Opt 46:1650-7, 2007). A fixed pulsedlaser diode was used as an excitation source at a wavelength of 670 nm.Region of interest (ROI) measurements with a spot size of 1.5 mm wereselected at the image plane. The laser power was automatically chosen asthe highest power that does not saturate the photon detector. Lifetimeanalysis was performed by using the ART OptiView (ART Advanced ResearchTechnologies, Inc.). Lifetime values and lifetime mapping werecalculated to fit fluorescence temporal point-spread functions (TPSFs)as single-exponential models with the Fit TPSF tool.

Photoimmunotherapy for In Vitro and In Vivo Models.

PIT was performed with a red light-emitting diode (LED) light at 680 to700 nm wavelength (Tech-LED, Marubeni America Co.) (Mitsunaga et al.,Bioconjug Chem. 23:604-9, 2012). Power densities were measured with anoptical power meter (PM 100, Thorlabs).

Determination of FLT for Pan-IR700.

Samples of Pan-IR700 at concentrations of 2.5, 5, 20, 40 pg/mL wereprepared by dilution with PBS. The fluorescence intensities andlifetimes of each sample were determined using the Optix MX2 system atroom temperature within a 1.7 ml centrifuge tube. To investigate theeffect of PIT using Pan-1R700, the FLT of each sample at theconcentration of 50 pg/mL was measured after irradiating the samples ata PIT dose of 0, 2, 4, 8, 15, 30 J/cm2.

Cell Line.

The HER1 positive cell line, A431 was used for HER1 targeting studieswith panitumumab conjugates. The cell line was grown in RPMI 1640 (LifeTechnologies) containing 10% fetal bovine serum (Life Technologies),0.03% L-glutamine, 100 units/mL penicillin, and 100 pg/mL streptomycinin 5% CO2 at 37° C.

Cell Pellet FLT Studies A431.

Cells were plated on 75 mm² cell culture flasks and incubated untilconfluent. Then Pan-IR700 conjugate was added to the media (1 pg/mL),and cells were incubated for 24 h at 37° C. Upon completion ofincubation, cells were removed from the flasks, and centrifuged toobtain pellets. The resulting cell pellets were washed with PBS×3 andplaced in 1.7 mL centrifuge tubes. The fluorescence intensities andlifetimes of each sample were then obtained. To investigate the effectof cellular internalization with Pan-IR700 conjugates, A431 cells wereplated on a 75 mm² flask and were incubated with Pan-1R700 for 1, 2, 4,6, 15 and 24 hours. After removing the flasks and obtaining A431 cellpellets, FLT measurements of the A431 pellet was acquired. After theA431 cell pellets were incubated overnight with Pan-R700, cell pelletswere irradiated at doses of 0, 2, 4, 8, 15, 30 J/cm2. After that, thesepellets were gently washed with PBS×1 and fluorescence intensity andlifetime images were obtained. To detect the antigen specificlocalization of IR700 and to confirm the morphological changes of A431cells before and after PIT, fluorescence microscopy was performed usingOlympus BX61 microscope (Olympus America) equipped with the followingfilters: a 590-650 nm excitation filter, a 665-740 nm band pass emissionfilter. Transmitted light differential interference contrast images(DIC) were also acquired. A431 cells were plated on a coverglass-bottomed culture well and incubated for 24 hours. Pan-IR700 wasadded to the medium (10 pg/mL), and the cells were incubated for either6 or 24 hours. Once complete, the cells were washed once with PBS, andfluorescence microscopy was performed before and after PIT.

Mouse Model.

A431 cells (HER1+, HER2−, 1×106 cells) were injected subcutaneously onboth sides of the dorsum of female nude mice (National Cancer InstituteAnimal Production Facility). The experiments were performed at 6-9 daysafter cell injection.

In Vivo FLT Imagine Studies after PIT.

Tumor-bearing mice were divided into 3 groups of 5 mice per group forthe following irradiation doses of PIT: 10, 30, and 50 J/cm². As acontrol, 5 mice were prepared without PIT. One hundred μg of Pan-IR700were injected intravenously via the tail vein into every mouse 24 hoursbefore PIT. A431 tumors in the right side of the dorsum were treatedwith PIT while the contralateral control tumors were shielded from lightexposure with aluminum foil. After PIT, FLT images were obtained at thefollowing time points: 0, 6, 24 and 48 hours. Zero hours acquisitionswere performed immediately after PIT. Maximum spot values of each ROI inthe FLT images were calculated for tumors on both sides of the dorsum.

Histological Analysis.

To evaluate serial histological changes immediately (within 5 min) afterPIT with various NIR light doses, microscopy was performed (BX51,Olympus America). A431 tumors were harvested in 10% formalin immediatelyafter 0, 10, 30, and 50 J/cm² of NIR light exposure. Serial 10-μm slicesections were fixed on a glass slide with H-E staining.

Statistical Analysis.

Statistical analyses were carried out using a statistics program(GraphPad Prism; GraphPad Software). Mann-Whitney's U test was used tocompare the lifetime value between those of treated tumors and untreatedtumors. Student's t test was used to compare with the lifetimes oftreated tumors to no treatment control. P<0.05 was considered toindicate a statistically significant difference.

Results

FLT is Independent from the Pan-IR700 Concentration in the Solution.

The FLTs of various concentrations of Pan-IR700 were approximately thesame, 3.56+/−0.081 ns; 3.62 (2.5 pg/mL), 3.58 (5 pg/mL), 3.44 (20pg/mL), 3.60 ns (40 pg/mL), whereas the fluorescence intensities weredecreased in proportion to the concentration (FIGS. 10A and 10B).

NIR Light Exposure Alone does not Affect the FLT of Pan-IR700.

Pan-IR700 (50 pg/mL) by itself was irradiated and FLT was measured. Bothfluorescence intensity and lifetime did not change by irradiation of LEDat the dose of 0, 2, 4, 8, 15, 30 J/cm2. The FLT was approximately3.44+/−0.058 ns.

Internalization of Pan-IR700 Prolonged the IR700 FLT.

FLT of A431 cells increased with the duration of the incubation withPan-1R700. The FLTs of A431 cell pellet at 1, 2, 4, 6 15 and 24 hours ofincubation were 2.98, 3.05, 3.13, 3.15, 3.36 and 3.4 ns, respectively.After 15 hours incubation, FLT of IR700 reached its peak and showed nofurther prolongation (FIG. 10D).

Greater Exposure of NIR Light Shortened the FLT of IR700 Containing A431Cells.

PIT with greater NIR light doses induced greater shortening of FLT inA431 cell pellets incubated with Pan-IR700 for 24 hours before exposureto the NIR light (FIG. 10C). PIT shortened the FLT of A431 pellets downto 3.28, 3.09, 2.94 and 2.85 ns at doses of 0, 8, 15 and 30 J/cm2,respectively.

PIT Induced Typical Necrotic Cell Death in A431 Cells as Well as Ruptureof Lysosomes.

Under microscopy, Pan-1R700 was seen on the cell membrane and withinendolysosomes at 24 hours after incubation. Following exposure to NIRlight, immediate damage was induced in the cell membranes and lysosomes.Multifocal bleb formation was seen in the cellular membranes,characteristic of necrotic cell death induced by PIT (FIG. 11).

Effective PIT Induced Immediate Shortening of the FLT of IR700 In Vivo.

The average FLT of A431 tumors 1 day after administration of 100 pg ofPan-IR700 in vivo was 3.27+/−0.46 ns (n=40). Significant shortening ofFLT was induced immediately after PIT with NIR light doses of 30 and 50J/cm² to experimental tumors (right dorsum, 30 J/cm²; down to61.5%+/−5.05% of untreated tumors in the same mouse; p<0.01, 50 J/cm²;down to 69.0%+110.92% of untreated tumors in the same mouse; p<0.05).

Transient prolongation of IR700 FLT was found in and around PIT treatedtumors 6 hours after PIT at NIR light doses of 30 and 50 J/cm2 butcontinued to shorten at >24 hours after PIT. PIT with 10 J/cm² did notshow this transiently prolonged FLT. IR700 FLT in untreated controltumors also slightly shortened at late time points (FIG. 12A).

Comparison with IR700 FLT between exposed and non-exposed tumors withNIR light of 30 and 50 J/cm² in the same mice showed significantdifferences at 0, 24 and 48 hours after PIT (p<0.05; FIGS. 12B and 12C).The differences of IR700 FLT at 6 hours post-PIT were not statisticallysignificant due to the diffuse temporal increase around exposed tumors.IR700 FLTs of exposed and non-exposed tumors with NIR light of 10 J/cm²did not show significant difference at any time point (FIG. 12D).

FLT in PIT treated tumors with 50 and 30 J/cm² shortened significantly(p<0.01) compared with no treatment controls (0 J/cm²). FLTs wereimmediately shortened to 69.1+/−10.9% and 61.5+/−5.1% by PIT with 50 and30 J/cm², respectively. A431 tumors irradiated with only 10 J/cm² showedno significant shortening of FLT immediately after PIT. FLT shortened byonly 7.7% at 48 hours after PIT compared with the untreated control(FIG. 13A). Interestingly, the FLT of non-irradiated tumors in PITtreated mice shortened slightly more than that in the untreated mice,but these changes were not significant, however, FLT became shorter withlarger doses of NIR light to the treated tumors (FIG. 13B). Thesechanges may be caused by small amounts of light diffusing through thesoft tissues from the “treated” side to the “untreated” side, thusexplaining the dose-dependence of the effect.

Histological Analysis.

Microscopy of treated tumors revealed various degrees of necrosis andmicro-hemorrhage with clusters of healthy or damaged but potentiallyviable tumor cells after PIT. Necrotic damage was diffuse and intenseand the amount of surviving tumor cells was reduced when 30 or 50 J/cm²of NIR light was administered. In contrast, when 10 J/cm² of NIR lightwas administered, necrotic cell damage was found in only limited areaswith relatively large areas of viable cancer cells accounting for themajority of the tissue (FIG. 13C).

Discussion

Fluorescence microscopy studies showed Pan-IR700 gradually internalizedinto lysosomes in A431 cells at 37° C. (FIG. 11). As Pan-IR700internalized (FIG. 10D) IR700 FLT became longer as a function ofincubation time. IR700 eventually accumulated in the lysosome. Afterexposure to a threshold intensity of NIR light, Pan-IR700 inducedimmediate outer cell membrane damage and damage to lysosomes resultingin accumulation of IR700 within the cytoplasm and into the extracellularspace. This damage was associated with a significant reduction in IR700FLT. This implies that cellular internalization of the Pan-IR700conjugate by itself prolongs IR700 FLT as it accumulates in theendolysosome. However, by damaging membrane structures, including thelysosomal membrane, PIT induces cell death and releases long FLT IR700,into the cytoplasm whereupon the FLT markedly shortens. Therefore,shortening FLT serves as an indicator of acute membrane damage inducedby PIT.

Treatment with PIT with effective therapeutic light dose of NIR leads toshortened IR700 FLT in cancer cells in vitro and in tumors in vivo. Theshortening of FLT was dependent on the dose of NIR light exposure invitro (FIG. 10C). PIT with suboptimal doses of NIR light (10 J/cm²) didnot show significant shortening of IR700 FLT in vivo. These differencescould be ascribed to the population of cancer cells, which received PITeffects. FIG. 7 demonstrates that PIT with 50 J/cm² of NIR lightexposure or more could eradicate A431 tumors. PIT with 30 J/cm² was notsufficient to totally eradicate tumors but caused tumor shrinkage andgrowth delay, indicating while not all cells were killed, most wereseverely and irreversibly damaged (Mitsunaga et al., Bioconjug. Chem.23:604-9, 2012).

Shortened FLT of treated tumor in vivo was observed within 30 minutes ofa single effective dose of NIR light and indicated a biologic effectseveral days before tumor size and shape changed. Although size of thelesion is considered a major indicator of cell death, it does not happenfast enough to determine if treatment has been effective. In thespecific case of PIT, where light can be reapplied if necessary, a moreimmediate readout of cell death is needed. Size changes do not occurrapidly enough for monitoring cytotoxic effects. This is especially trueof surgical or endoscopic procedures where it is preferable to completetreatments at one setting (Mitsunaga et al., Bioconjug. Chem. 23:604-9,2012). FLT, because it is an immediate readout of the tumor's condition,can assess the therapeutic effects of PIT to the cancer cellsimmediately after treatment and aids in deciding whether additionaldoses of NIR light exposure are necessary or not during the procedure(Kosaka et al., Int J Cancer 129:1671-7, 2011; Longmire et al., CancerSci 100:1099-104, 2009).

Interestingly, after an initial shortening of the FLT, it briefly becamelonger at about 6 hours after PIT. By 24 hours after PIT the FLT wasreduced again (FIG. 12). Since prolongation of IR700 FLT as it is beinginternalized was observed, it is proposed that after cell membranedisruption caused by PIT, the IR700 leaks into the extracellular spacewhere it is internalized by macrophages mobilized to respond to therelease of cytokines associated with cell necrosis. This is supported byhistologic findings at 6 hr post-PIT that show inflammatory infiltratescomposed of macrophages, which are entering the space formerly occupiedby viable tumor (Mitsunaga et al., Nat Med 17:1685-91, 2011). Thistransient prolongation of IR700 FLT may therefore be a sign of effectivecell damage followed by initiating tissue repair possibly mediated bythe chemokine release or the toll-like receptor system induced by thefragmented DNA and lipid bilayer (Emeagi et al., Cancer Res 72:1342-52,2012; Shiratsuchi et al., J Immunol 172:2039-47, 2004; Zhu et al., Cell24:615-29, 2006).

Fluorescent proteins (FPs) are a potential alternative for monitoringtumor growth in vivo (Kimura et al., J Cell Biochem 110:1439-46, 2010;Tsai et al., Anticancer Res 30:3291-4, 2010; Yamamoto et al., Cancer Res64:4251-6, 2004; Hoffman and Yang, Nat Protoc 1:1429-38, 2006).Fluorescence imaging using FPs is better suited for longitudinalmonitoring of the effects of photo-therapy (Jiang et al., Cell Cycle5:1198-201, 2006; Hoffman and Yang, Nat Protoc 1:775-82, 2006). Acutely,FPs retain their signal regardless of the viability of the cells andeven in necrotic cells may be taken up by macrophages. Thus, even thoughFLT requires post-processing of the fluorescence signal and usesrelatively expensive equipment, it is better suited for detecting acutechanges than FPs (Hoffman and Yang, Nat Protoc 1:928-35, 2006; Hoffman,Nat Rev Cancer 5:796-806, 2005). Fluorescence imaging with FPs has beenused for longitudinal monitoring of the therapeutic effects of PIT(Mitsunaga et al., Bioconjug Chem 23:604-9, 2012). However, PIT-inducedacute cell death can only be detected with optical methods such as FLTwhile longer term changes can be measured with FPs. FLT is clinicallytranslatable while FPs, which require cell transfection, are unlikely tobe used clinically.

This data demonstrates that the FLT of Pan-IR700 is a robust measurementthat does not depend on the concentration of Pan-IR700 or light exposurein solution. For example, the in vitro Pan-IR700 solution did not changeits FLT at varying concentrations or after NIR light exposure withvarious doses. Therefore, only the surrounding chemical microenvironmentseems to affect the IR700 FLT. While IR700 is normally fluorescent andreflects tumor burden, after catabolism in the lysosome andphoto-bleaching, fluorescence may be reduced, thus leading to ambiguityregarding tissue viability. However, those photo-chemical andbiochemical changes do not affect FLT. Therefore, shortening of FLT is abetter biomarker than IR700 fluorescence intensity.

In conclusion, FLT can be used for assessing in near-real-time, thecytotoxic effects of PIT employing a mAb-IR700 conjugate, duringsurgical or endoscopic procedures. FLT is prolonged during endolysomalinternalization but rapidly shortened after cell damage. FLT again isprolonged for a brief period about 6 hours after PIT due tointernalization by migrating macrophages. After that there is a steadyreduction in FLT. Thus, FLT imaging thus, allows the assessment of theeffect of PIT before morphological changes become evident.

Example 15 Combination Photoimmunotherapy and Chemotherapy

This example describes methods used to combine PIT with other therapiesfor cancer treatment, such as chemotherapy. It is demonstrated that theenhanced permeability generated during PIT enhances delivery ofnano-sized agents.

Materials and Methods

Cells.

A431 cells expressing HER1 were used for PIT. Cells were grown inRPMIl640 supplemented with 10% FBS and 1% penicillin-streptomycin intissue culture flasks in a humidified incubator at 37° C. in anatmosphere of 95% air and 5% carbon dioxide.

Reagents.

IRDye 700DX NHS ester (IR700; C₇₄H₉₆N₁₂Na₄O₂S₆Si₃, molecular weight of1954.22) and IRDye 800CW NHS ester (IR800; C₅₀H₅₄N₃Na₃O₁₇S₄, molecularweight of 1166.20) were from LI-COR Bioscience. Panitumumab, a fullyhumanized IgG2 monoclonal antibody (mAb) directed against the humanepidermal growth factor receptor (EGFR; HER1) was from Amgen.Trastuzumab, a recombinant humanized mAb directed against the humanEGFR2 (HER2) was from Genentech. Qtracker 800 non-targeted quantum dotswas from Invitrogen. All other chemicals used were of reagent grade.

Synthesis of IR700 and IR800-Conjugated mAbs.

Conjugation of dyes with mAbs was performed according to the procedurereported in the examples above. Each mAb (1 mg, 6.8 nmol) was incubatedwith IR700 (60.2 μg, 30.8 nmol) or IR800 (35.9 μg, 30.8 nmol) in 0.1 moll⁻¹ Na2HPO₄ (pH 8.6) at room temperature for 1 h. The mixture waspurified with a Sephadex G50 column (PD-10; GE Healthcare). Theconcentration of dye and protein was measured by absorption with thespectroscopy (8453 Value System; Agilent Technologies) to confirm thenumber of fluorophore molecules conjugated to each mAb molecule.

In Vivo Nanodrug-Delivery after Photoimmunotherapy.

Six-week-old to 8-week-old female homozygote athymic nude mice werepurchased from Charles River (National Cancer Institute Frederick).During treatment, mice were anesthetized with isoflurane. Two millionA431 cells were injected subcutaneously in the right and left dorsums ofeach mouse. Five days after cell injection, 100 μg of Pan-IR700 wasadministered intravenously, and 1 day later, either side of tumor wasirradiated with NIR light from a red-light-emitting diode at wavelengthsof 670-690 nm and a power density of 10-100 J cm⁻², as measured with anoptical power meter (PM 100 (Thorlabs)). One hour after PIT, Pan-IR800(100 μg), Qtracker 800 Non-Targeted Quantum dots (32.5 pmol), orDaunoXome (30 mg kg⁻¹) were injected intravenously, and the in vivofluorescence images were obtained with a Pearl Imager (LI-CORBiosciences) and a Maestro Imager (CRi). For MR imaging, SPIO (Feridex)was administered intravenously 1 h post-PIT and MR images were obtained.The tumors were excised, and frozen or paraffin-embedded forhistological study and fluorescence microscopy study after ex vivoimaging.

In Vivo Fluorescence Imaging.

Five days after injection of two million A431 or 3T3-HER2 cells in rightand left dorsums, and seven days after MDA-MB-468 cells injection inmammary pads, tumor volumes of approximately 75 mm³ were selected.Signals of IR700 and IR800 was detected with a fluorescence camera(Pearl Imager, LI-COR Biosciences) using the 700 and 800 nm fluorescencechannel. Qdot800 was detected with Maestro in vivo Imaging System (CRi)using a band-pass filter, which ranges between 575 to 605 nm(excitation) and a long-pass NIR filter over 800 nm (emission).Fluorescence images of daunorubicin were also obtained with Maestrousing a band-pass filter from 503 to 555 nm (excitation) and a long-passgreen filter over 580 nm (emission). The tunable emission filter wasautomatically stepped in 10 nm increments from 650 to 950 nm and from500 to 800 nm for the NIR and green filter sets at constant exposure.The spectral fluorescence images consist of autofluorescence spectra andthe spectra from Qdot800 and daunorubicin, which were then unmixed,based on their spectral patterns using commercial software (Maestrosoftware; CRi). Mice were sacrificed with carbon dioxide immediatelyafter in vivo imaging. The tumors were excised, and frozen orparaffin-embedded for histological study and fluorescence microscopystudy after ex vivo imaging.

Therapeutic studies.

To determine the effectiveness of super EPR effect for tumor therapy,whether PIT could enhance the therapeutic effect of DaunoXome wasinvestigated as follows. One million A431 cells were injectedsubcutaneously in the right dorsum of the mice. To determine the tumorvolume, the greatest longitudinal diameter (length) and the greatesttransverse diameter (width) were determined with an external caliper.Tumor volume based on caliper measurements was calculated by thefollowing formula; tumor volume=length×width×0.5. Tumors reachingapproximately 40 mm³ in volume were selected. Selected mice wererandomized into 4 groups of at least 10 mice per group for the followingtreatments: (1) no treatment; (2) DaunoXome (6 mg kg⁻¹); (3) PIT (50 Jcm⁻²); (4) PIT (50 J cm⁻²), 1 h later, DaunoXome (6 mg kg⁻¹). Aftertreatment, the mice were monitored daily and their tumor volume wasmeasured twice a week until it reached 750 mm³, at which time mice wereeuthanized with carbon dioxide gas.

Fluorescence Microscopy.

Ten-μm-thick frozen or paraffin sections were prepared and fluorescencein tumor sections was detected using an Olympus BX81 microscope (OlympusAmerica, Inc., Melville, N.Y.) equipped with the following filters:excitation wavelength 590 to 650 nm, and 480 to 550 nm, emissionwavelength 662.5 to 747.5 nm, 765 to 855 nm, and 590 nm long pass forIR700, Qdot800, and daunorubicin, respectively. Transmitted lightdifferential interference contrast images were also acquired. H&Estaining and Prussian Blue staining were performed according to standardprotocol.

Optimal Timing of Second Shot.

To determine the optimal timing of second shot, Pan-IR800 (100 μg) wasadministered intravenously into A431 bearing mice at 1, 6, and 24 hafter PIT treatment, and the dynamic imaging for 1 h was carried outwith Pearl Imager according to the protocol described above.

Statistical Analysis.

Data are expressed as means+s.e.m. from a minimum of three experiments,unless otherwise indicated. Statistical analyses were carried out usinga statistics program (GraphPad Prism; GraphPad Software). For multiplecomparisons, a one-way analysis of variance (ANOVA) with post test(Kruskal-Wallis test with post-test) was used. The cumulativeprobability of survival, determining herein as the tumor volume wasfailed to reach 750 mm³, were estimated in each group with the use ofthe Kaplan-Meier survival curve analysis, and the results were comparedwith use of the log-rank test with Bonferroni's correction formultiplicity. P<0.05 was considered to indicate a statisticallysignificant difference.

Results Pit Increases the Perfusion in the Tumors

To validate the change of perfusion in the tumors after PIT treatment,dynamic distribution of PEGylated quantum dot 800 (non-targeted Qdot800)was evaluated in A431 (HER1 positive) bearing mice. A431 tumors weretreated with a single dose of NIR light (50 J cm⁻²) at 1 d afterinjection of IR700 conjugated anti-HER 1 mAb (panitumumab) (Pan-IR700).Qdot800 was administered 1 h after light irradiation, and in vivodynamic imaging studies were carried out. The diameter of Qdot800 was anaverage of 50 nm, which was determined by size-exclusion HPLC andSDS-PAGE. Rapid accumulation of Qdot800 was observed in the PIT-treatedtumors within 1 h, while no significant uptake was detected in thecontrol tumor without exposure to NIR light (FIG. 14A). The increasingrate of signal intensity (SI) between 1 min and 60 min was 25.7-foldhigher in the PIT-treated tumor than in control tumor, which wascalculated by the following equation as a super EPR index:[(SI_(PIT at 60 min)−SI_(Back at 60 min))−(SI_(PIT at 1 min)−SI_(Back at 1 min))]/[(SI_(Control at 60 min)−SI_(Back at 60 min))−(SI_(Control at 1 min)−SI_(Back at 1 min))](FIG. 14B). The signals in PIT-treated tumors were highly maintained by24 h, indicating the long-term retention of Qdot00.

A pathological analyses and fluorescence microscopic studies revealedthat PIT resulted in necrotic damages of tumor cells and that Qdot800was broadly distributed in the necrotic regions and interstitium in thePIT-treated tumors (FIG. 14C). CD31 staining demonstrated that most ofblood vessels in the tumors were decrepit and surrounding tumor cellswere also severely damaged (FIG. 14C). On the other hand, A431 cellswere almost alive in the control tumors and the fluorescence signals ofQdot800 were focal in the vicinity of main blood vessels (FIG. 14C).

Clinical-use magnetic resonance imaging (MRI) contrast agent, superparamagnetic iron oxide (SPIO) (diameter 200 nm), was challenged todetermine the breakpoint of permeability. Within 5 min after injectionof SPIO, the signal intensity in the PIT-treated tumors was dramaticallyreduced, while slight decrease in the control tumors were observed (FIG.15A). The decreasing rate of signal intensities at 60 min was higher inthe PIT-treated tumor than in control tumor. SPIO was accumulated in thenecrotic regions and interstitium in the PIT-treated tumors, which wasconfirmed with Prussian Blue staining (FIG. 15B). Similar results wereobtained with gadolinium (Gd) labeled polyamidoamine dendrimer(generation 6t) (G6-Gd) and USPIO as T1 and T2 contrast agents,respectively. G6-Gd (diameter 10 nm) was distributed streakly inPIT-treated tumors with time, in contrast, only main vessels on thetumor surfaces were intensely described in the control tumors.

USPIO (diameter 30 nm) was also rapidly taken up by PIT-treated tumorsespecially in the interstitium and necrotic regions as suggested byPrussian Blue staining. These results demonstrate that nanoparticleswith at least 200 nm of diameter exhibit massive and rapid leakage intotumor tissues treated by PIT, and thus this super EPR method can beapplied to cancer therapy.

PIT Enhances the Delivery and Efficacy of Anti-Cancer Drugs

The super EPR regimen was tested with second shot of panitumumab(diameter 10 nm) in A431 bearing mice. Panitumumab is a therapeuticmonoclonal antibody in clinical use for the treatment ofEGRR-expressing, metastatic colorectal carcinoma. The usefulness ofpanitumumab has also been validated in breast, lung, head, and neckcancers. PIT facilitated the permeability of Pan-IR800 in treated tumorswithin 10-60 min, while no change on signal intensities were detected incontrol tumors, consistent with nanoparticles including Qdot800 andG6-Gd (FIG. 16A). The effective light dose needed to achieve sufficientdelivery of anti-cancer drugs (Pan-IR800) was determined. Signalintensities of IR800 in PIT-treated tumors increased with time in alight dose-dependent manner (FIG. 16B), and super EPR index of Pan-IR800in PIT-treated tumors were significantly higher than in control tumorsbetween 1 min and 60 min after probe injection. Slight increase ofsignal intensity was observed in the control tumor in groups irradiatedby high dose of NIR light, probably because scattered NIR light crossedover from the irradiated side (FIG. 16A). A pathological study revealedthat necrotic cell death in PIT-treated tumors was more intense whenexposed to high dose NIR light. Interestingly, the change of perfusionby PIT was gradually vanished with time after treatment, and wascompletely stopped within 24 h (FIGS. 16 C and 16D), indicating therepair of the barrier between blood vessels and tumor tissues or thecomplete blockage of blood flow. These results indicated that optimaltiming of second shot is by 6 h. On the basis of the similarity of thechange of perfusion in the tumors after PIT induced with two differentmAbs (panitumumab and trastzumab) against three different cells (A431(HER1 positive), 3T3-HER2 (HER2 positive), and MDA-MB-468 (HER1positive)) expressing various numbers of respective target molecules,thus this super EPR is generally applicable to other mAbs and antigens(FIG. 18).

To examine the potential of molecular non-targeted therapeutic agentsfor efficient cancer therapy based on super EPR effect, liposomecontaining daunorubicin (DaunoXome; DX) (diameter 50 nm) was scanned andapplied therapeutic studies. DX was rapidly accumulated and retained inPIT-treated tumors for 1 h like SPIO and USPIO (FIGS. 17A and 17B). Theincreasing rate of signal intensities at 60 min was higher in thePIT-treated tumor compared with in control tumor (FIG. 17C). Similar toQdot800, DX was widely distributed encircling the survived tumor tissuesin PIT-treated tumors, and the colocalization of IR700 (indicatingsurvived tumor cells) and DX was partially observed, whereas, thesignals of DX in control tumors were localized in the vicinity of mainblood vessels (FIG. 17D). This phenomenon was demonstrated both in themargin and core of tumors. A431 tumors were treated with a single doseof light (50 J cm⁻²) at 1 d after injection of Pan-IR700. The efficacyof the methods was determined in four groups of A431 bearing mice (n≥10in each group). All the tumors we treated had an area of less than 750mm³, as larger tumors were associated with side effects (subcutaneousbleeding, tumor bleeding, or a weakened state) that, in accordance withour institution's animal care and use guidelines, required that the micebe we euthanized. Tumor volume was significantly reduced in A431 tumorswith combination therapy of PIT and DX compared to untreated controlmice, mice treated with DX only and PIT only (FIG. 17E), and survivalwas significantly prolonged in mice with combination therapy of PIT andDX than other groups (FIG. 17F). No obvious loss of body weight wasobserved in the PIT plus DX group.

In another experiment, 3T3/HER2 mice were injected with Tra-IR700, and24 h later, NIR light (50 J/cm2) were irradiated to the right sidetumor. Tra-IR800 was administered 1 h after PIT treatment. As shown inFIG. 19A, only the right sided tumor was clearly shown up within 10 min.Thus, Tra-IR800 can be accumulated in only the regions where the tumorwas exposed to NIR light. In another experiment, MDA-MB-468 bearing micewere injected with Pan-IR700, and 24 h later, NIR light (50 J/cm2) wereirradiated to the right side tumor. Pan-IR800 was administered 1 h afterPIT treatment. As shown in FIG. 19B, only the right sided tumor wasclearly shown up within 10 min.

A431 mice were injected with Pan-IR700, and 24 h later, NIR light (50J/cm2) were irradiated to the right side tumor. USPIO was administered 1h after PIT treatment. As shown in FIG. 20A, only the right sided tumorwas clearly shown up within 5 min. Prucian blue staining and HE stainingis shown in FIG. 20B. The dynamic images of G6-Gd after PIT. A431 micewere injected with Pan-IR700, and 24 h later, NIR light (50 J/cm2) wereirradiated to the right side tumor. G6-Gd was administered 1 h after PITtreatment. As shown in FIG. 20C, only the right sided tumor was clearlyshown up within 5 min.

Fluorescence microscopic studies in the margin and core regions oftumors was determined. IR700 signal in FIG. 21A shows the A431 cellsthat survived. Daunorubicin containing liposome was broadly distributedin the PIT-treated tumor tissues and co-localization of IR700 andDaunorubicin containing liposome was partially observed. Especially inthe core regions, DX can be taken up in the locally necrotic regions.FIG. 21B shows the change of body weight after therapy. There was noobvious difference between groups.

In conclusion, antibody-IR700 PIT therapy was effective, only when theantibody conjugates were bound to the cell membrane, but showed nophototoxicity when they were not bound or irradiated with NIR light. Inaddition, PIT induced super enhanced permeability effects (Super-EPReffect) which helped the delivery of nano-sized agents. Thus, PIT usingMAb-IR700 combination with nano-sized agents can be used fortheranostics for highly selective and effective treatment of cancers.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that illustratedembodiments are only examples of the disclosure and should not beconsidered a limitation on the scope of the invention. Rather, the scopeof the invention is defined by the following claims. We therefore claimas our invention all that comes within the scope and spirit of theseclaims.

We claim:
 1. A phototoxic pharmaceutical composition for treating atumor, comprising: a phototoxic conjugate comprising an IR700 moleculeconjugated to an antibody that binds HER1; and a pharmaceutical carrier,wherein the phototoxic conjugate exhibits phototoxicity to kill tumorcells.
 2. The pharmaceutical composition of claim 1, wherein theantibody is selected from the group consisting of cetuximab,panitumumab, zalutumumab, nimotuzumab and matuzumab, or an antigenbinding fragment thereof.
 3. The pharmaceutical composition of claim 1,wherein the antibody is cetuximab.
 4. The pharmaceutical composition ofclaim 1, wherein the antibody is panitumumab.
 5. The pharmaceuticalcomposition of claim 1, wherein the antibody is zalutumumab.
 6. Thepharmaceutical composition of claim 1, wherein the antibody isnimotuzumab.
 7. The pharmaceutical composition of claim 1, wherein theantibody is matuzumab.
 8. A combination, comprising: a first compositioncomprising a phototoxic conjugate comprising an IR700 moleculeconjugated to an antibody that binds HER1; and a second compositioncomprising an additional therapeutic agent.
 9. The combination of claim8, wherein the first and/or second composition comprise apharmaceutically acceptable carrier.
 10. The combination of claim 8,wherein the antibody is selected from the group consisting of cetuximab,panitumumab, zalutumumab, nimotuzumab and matuzumab, or an antigenbinding fragment thereof.
 11. The combination of claim 8, wherein theantibody is cetuximab.
 12. The combination of claim 8, wherein theantibody is panitumumab.
 13. The combination of claim 8, wherein theantibody is zalutumumab.
 14. The combination of claim 8, wherein theantibody is nimotuzumab.
 15. The combination of claim 8, wherein theantibody is matuzumab.
 16. The combination of claim 8, wherein the firstand second composition are formulated separately.
 17. The combination ofclaim 8, wherein the therapeutic agent is an anti-cancer oranti-neoplastic agent.
 18. The combination of claim 8, wherein thetherapeutic agent is selected from the group consisting of aradiotherapeutic agent, a chemotherapeutic agent, an antibiotic, analkylating agent, an antioxidant and a kinase inhibitor.
 19. Thecombination of claim 8, wherein the therapeutic agent is selected fromamong a microtubule binding agent, a DNA intercalator or cross-linker, aDNA synthesis inhibitor, a DNA or RNA transcription inhibitor, anantibody, an enzyme, an enzyme inhibitor and a compound that affectsgene regulation.
 20. The combination of claim 8, wherein the therapeuticagent is selected from among paclitaxel, docetaxel, vinblastine,vindesine, vinorelbine, epothilone, colchicine, dolastatin 15,nocodazole, podophyllotoxin, rhizoxin, actinomycin D, daunorubicin,cisplatin, carboplatin, oxaliplatin, mitomycin C, bleomycin,chlorambucil, cyclophosphamide, methotrexate, 5-fluro-5′-deoxyuridine,5-fluorouracil, camptothecin, etoposide, formestane, trichostatin,raloxifene, 5-azacytidine, 5-aza-2′-deoxycytidine, tamoxifen,4-hydroxytamoxifen, mifepristone, Gleevac, Iressa, Tarceva, adriamycine,apigenin, rapamycine, zebularine, cimetidine, interleukin-2 (IL-2), andan anti-CTLA4 antibody or analogs or derivatives thereof.